Methods for producing polypeptide binding sites, monoclonal antibodies and compositions thereof

ABSTRACT

The present invention describes methods for producing binding sites on polypeptides, and particularly for producing binding sites within the CDR regions of immunoglobulin heavy or light chains that are displayed on the surface of filamentous phage particles. The invention also describes oligonucleotides useful for preparing the binding sites, and human monoclonal antibodies produced by the present methods.

This application is a 371 of PCT/US94/01258 filed Feb. 2, 1994, which isa continuation-in-part of Ser. No. 08/084,542 filed Jun. 28, 1993 nowabandoned, which is a continuation-in-part of Ser. No. 08/012,566 filedFeb. 2, 1993 now abandoned.

This invention was made with government support under Contract No. 1RO1CA56483 by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates generally to the field of proteinbiochemistry and immunology, and relates specifically to methods forproducing polypeptide binding sites specific for preselected targets.

BACKGROUND

Protein interactions with other molecules is basic to biochemistry.Protein interactions include receptor-ligand interactions,antibody-antigen interactions, cell-cell contact and pathogeninteractions with target tissues. Protein interactions can involvecontact with other proteins, with carbohydrates, oligosaccharides,lipids, metal ions and the like materials.

The basic unit of protein interaction is the region of the proteininvolved in contact and recognition, and is referred to as the bindingsite.

There is an increasing need to find new molecules which can effectivelymodulate a wide range of biological processes, for applications inmedicine and agriculture. Thus, there is a need for systematic and rapiddevelopment of binding sites on proteins for use in the construction ofprotein binding site analogs and antagonists, proteins with improved oraltered binding specificities and the attendant altered functionassociated with the altered specificity, and antibodies with uniqueantigen specificities.

Numerous strategies have been developed for preparing proteins havingnew binding specificities besides the conventional technique of randomscreening of natural products. These approaches generally involve thesynthetic production of large numbers of random molecules followed bysome selection procedure to identify the molecule of interest. Forexample, epitope libraries have been developed using random polypeptidesdisplayed on the surface of filamentous phage particles. The library ismade by synthesizing a repertoire of random oligonucleotides to generateall combinations, followed by their insertion into a phage vector. Eachof the sequences is separately cloned and expressed in phage, and therelevant expressed peptide can be selected by finding those phage thatbind to the particular target. The phages recovered in this way can beamplified and the selection repeated. The sequence of the peptide isdecoded by sequencing the DNA. See for example Cwirla et al., Proc.Natl. Acad. Sci., USA, 87:6378-6382 (1990); Scott et al., Science,249:386-390 (1990); and Devlin et al., Science, 249:404-406 (1990).

Another approach involves large arrays of peptides that are synthesizedin parallel and screened with acceptor molecules labelled withfluorescent or other reporter groups. The sequence of any effectivepeptide can be decoded from its address in the array. See for exampleGeysen et al., Proc. Natl. Acad. Sci., USA, 81:3998-4002 (1984); Maejiet al., J. Immunol. Met., 146:83-90 (1992); and Fodor et al., Science,251: 767-775 (1991).

In another approach, Lam et al., Nature, 354:82-84 (1991) describescombinatorial libraries of peptides that are synthesized on resin beadssuch that each resin bead contains about 20 pmoles of the same peptide.The beads are screened with labelled acceptor molecules and those withbound acceptor are searched for by visual inspection, physicallyremoved, and the peptide identified by direct sequence analysis. Inprinciple, this method could be used with other chemical entities but itrequires sensitive methods for sequence determination.

A different method of solving the problem of identification in acombinatorial peptide library is used by Houghten et al., Nature,354:84-86 (1991). For hexapeptides of the 20 natural amino acids, 400separate libraries are synthesized, each with the first two amino acidsfixed and the remaining four positions occupied by all possiblecombinations. An assay, based on competition for binding or otheractivity, is then used to find the library with an active peptide. Thentwenty new libraries are synthesized and assayed to determine theeffective amino acid in the third position, and the process isreiterated in this fashion until the active hexapeptide is defined. Thisis analogous to the method used in searching a dictionary; the peptideis decoded by construction using a series of sieves or buckets and thismakes the search logarithmic.

Large libraries of wholly or partially synthetic antibody combiningsites, or paratopes, have been constructed utilizing filamentous phagedisplay vectors, referred to as phagemids, yielding large libraries ofmonoclonal antibodies having diverse and novel immunospecificities. Thetechnology uses a filamentous phage coat protein membrane anchor domainas a means for linking gene-product and gene during the assembly stageof filamentous phage replication, and has been used for the cloning andexpression of antibodies from combinatorial libraries. Kang et al.,Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991). Combinatoriallibraries of antibodies have been produced using both the cpVIIImembrane anchor (Kang et al., supra) and the cpIII membrane anchor.Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991).

The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chain genes(Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123, 1991), byaltering the CDR3 regions of the cloned heavy chain genes of the library(Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461, 1992), and byintroducing random mutations into the library by error-prone polymerasechain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA,89:3576-3580, 1992).

Mutagenesis of proteins has been utilized to alter the function, and insome cases the binding specificity, of a protein. Typically, themutagenesis is site-directed, and therefore laborious depending on thesystematic choice of mutation to induce in the protein. See, for exampleCorey et al., J. Amer. Chem. Soc., 114:1784-1790 (1992), in which rattrypsins were modified by site-directed mutagenesis. Partialrandomization of selected codons in the thymidine kinase (TK) gene wasused as a mutagenesis procedure to develop variant TK proteins. Munir etal., J. Biol. Chem., 267:6584-6589 (1992).

Using the random synthetic hexapeptide library displayed on filamentousphage, O'Neil et al., Science, 249:774-778 (1990), described theidentification of a variety of different hexapeptides that contain thesequence Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) and that bind to theintegrin GPIIb/IIIa.

In another approach, Roberts et al., Gene, 121:9-15 (1992), describesthe point mutation of a protease inhibitor (BPTI) as a fusion proteinwith gene III of a phagemid, and demonstrated a change in bindingspecificity such that the mutant binds human neutrophil elastase ratherthan trypsin. Similarly, Roberts et al., Proc. Natl. Acad. Sci., USA,89:2429-2433 (1992), produced by mutagenesis a library of phagedisplaying mutant trypsin inhibitor, and isolated variant enzymes withincreased affinity.

Tomiyama described an antibody designated PAC-1 which binds the integrinGPIIb-IIIa and contains the sequence Arg-Tyr-Asp (RYD) in the antibody'sthird complementarity determining region of the heavy chain. Tomiyama etal., J. Biol. Chem., 267:18085-18092 (1992). Antibody PAC-1 is a markerfor platelet activation and its binding to GPIIb-IIIa can be blockedusing peptides that contain the RGD sequence.

BRIEF DESCRIPTION OF THE INVENTION

Methods have now been discovered using the phagemid vectors to produce abinding site capable of binding (interacting with) any of a largevariety of target molecules.

Thus, in one embodiment, the invention describes a method for producingin a polypeptide a binding site capable of binding a preselected agentcomprising introducing a nucleotide sequence that codes for an aminoacid residue sequence defining the binding site into a CDR region of anucleic acid comprising an immunoglobulin heavy or light chain gene byamplifying the CDR region of the immunoglobulin gene by a primerextension reaction using a primer oligonucleotide. The oligonucleotidehas 5′ and 3′ termini and comprises:

i) a nucleotide sequence at the 3′ termini capable of hybridizing to afirst framework region of the immunoglobulin gene;

ii) a nucleotide sequence at the 5′ termini capable of hybridizing to asecond framework region of the immunoglobulin gene; and

iii) a nucleotide sequence between the 5′ and 3′ termini according tothe formula:—X—[MNN]_(a)—Y—[MNN]_(b)—X—,where the sum of a and b is from 5 to 50, X is a trinucleotide encodingcysteine or a native amino acid residue coded by the immunoglobulingene, N is independently any nucleotide, M is adenine (A) or cytosine(C) or analogs thereof, Y is a nucleotide sequence that encodes aminimum recognition domain of the binding site, and the 5′ and 3′terminal nucleotide sequences have a length of about 6 to 50 nucleotidesin length.

In preferred embodiments, the immunoglobulin is human, and morepreferably the CDR is CDR3.

Additionally contemplated for use in this invention is the complementarynucleotide sequence of the preferred oligonucleotide formulation andidentified sequences. Specifically, the complementary nucleotide has 5′and 3′ termini between which is the nucleotide formulation—X—[NNK]_(a)—Y—[NNK]_(b)—X—. This alternative embodiment of acomplementary oligonucleotide thus hybridizes to the non-coding(antisense) strand of the template DNA.

Insofar as the method can be applied to any of a variety of knownprotein binding sites, the binding site can be an RGD-dependent bindingsite, a CD4 receptor binding site on HIV gp120, or a vitronectinreceptor binding site on vitronectin to name but a few possibilities.

Also contemplated is an oligonucleotide useful as a primer for producinga binding site in a polypeptide coded for by a immunoglobulin heavy orlight chain gene. The oligonucleotide has 5′ and 3′ termini andcomprises:

i) a nucleotide sequence at the 3′ termini capable of hybridizing to afirst framework region of the immunoglobulin gene;

ii) a nucleotide sequence at the 5′ termini capable of hybridizing to asecond framework region of the immunoglobulin gene; and

iii) a nucleotide sequence between the 5′ and 3′ termini according tothe formula:—X—[MNN]_(a)—Y—[MNN]_(b)—X—,where the sum of a and b is from 5 to 50, X is a trinucleotide encodingcysteine or a native amino acid residue coded by the immunoglobulingene, N is independently any nucleotide, M is adenine (A) or cytosine(C) or analogs thereof, Y is a nucleotide sequence that encodes aminimum recognition domain of the binding site, the 5′ and 3′ terminalnucleotide sequences have a length of about 6 to 50 nucleotides inlength, and sequences complementary thereto.

In preferred embodiments, the oligonucleotide hybridizes to a humanimmunoglobulin, and more preferably defines a heavy chain CDR3 domain.

In another embodiment, the invention describes human monoclonalantibodies produced by the methods of the invention.

Preferred antibodies bind platelet glycoprotein gpIIb/IIIa, and inhibitplatelet aggregation. Other antibodies described herein exhibit bindingspecificity for one and not other vitronectin receptor molecules.Particularly preferred antibodies immunoreact with target with anaffinity of at least 1×10⁻⁹ molar (M).

Polypeptides are described which inhibit fibrinogen binding togpIIb/IIIa, and are also useful in therapeutic methods for inhibitingplatelet aggregation. Further polypeptides are described which inhibitvitronectin binding to vitronectin receptor α_(v)β₃, and are also usefulin therapeutic methods for inhibiting vitronectin binding to vitronectinreceptor α_(v)β₃.

Therapeutic compositions containing the monoclonal antibodies orpolypeptides of this invention are also described.

DNA expression vectors capable of expressing a phagemid immunoglobulindisplay protein are described which comprise a polynucleotide sequencethat codes an immunoglobulin heavy chain polypeptide that includes inthe CDR3 portion of the heavy chain a binding site able to bind apreselected target molecule.

Other embodiments will be apparent to one skilled in the art in view ofthe teachings within.

A major advantage of the methods of the invention derives from the factthat new binding specificities can be rapidly developed for use in avariety of ways, including the development of ligands with alteredspecificity for receptor, receptors with altered specificity for ligand,altered and/or increased antibody binding specificity, and new proteinsor polypeptides that can bind oligosaccharides, nucleic acids, and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates the engineering and selection of syntheticanti-integrin antibodies as taught by the methods of this invention.Many RGD motifs are displayed at the apex of a flexible loop. Sinceantibody CDR's, particularly HCDR3, often exist as flexible loops, anintegrin recognition sequence into this region. To optimize antibodybinding affinity a phage library was constructed in which the residuesflanking the RGD were randomized. The resulting antibodies wereexpressed as a fusion protein on the surface of bacteriophage. Theoptimal antibody from the initial selection, Fab 9, bound α_(v)β₃ andα_(IIb)β₃ with high affinity. A similar strategy was used to obtainspecificity for platelet integrin α_(IIb)β₃ and to identify inhibitorymotifs that lack RGD. The sequences flanking the RGDI from Fab 9 weremaintained to preserve the optimal presentation, but positions 5-8 wererandomized in a new phage library termed MTF. Antibodies specific forα_(IIb)β₃ were selected from this library by competing with purifiedα_(v)β₃ in solution and subsequently panning on purified α_(IIb)β₃. Thesequences shown in FIG. 1 are VGCXXXRGDXXXCYY (positions 6-20 of SEQ IDNO:56), VGCSFGRGDIRNCYY (positions 1-15 of SEQ ID NO:4), andVGCSFGXXXXRNCYY (positions 1-15 of SEQ ID NO:67).

FIG. 2 illustrates a schematic of a phagemid surface display vector usedin the invention as described in Example 2, indicating the relationshipbetween the display vector and manipulations to form a vector forproducing soluble Fab fragments. The sequences shown in FIG. 2 are AOVKL(SEQ ID NO:77) and GGGGS (SEQ ID NO:78).

FIGS. 3A and 3B illustrate the motif optimization of Fab 9 forgenerating antibodies with specificity for α_(IIb)β₃. Antibodies withbinding preference for α_(IIb)β₃ were generated by re-design of Fab 9 asdescribed in Example 1D. Following selection by panning, and initialscreening by ELISA, the MTF antibodies were tested in ligand inhibitionstudies as described in Example 5. The ability of two antibodies, MTF-2shown in FIG. 3A and MTF-10 shown in FIG. 3B to block ligand binding isshown. Ligand binding to α_(v)β₃ as indicated by the lines marked withfilled circles was measured with ¹²⁵I-vitronectin and ligand binding toα_(v)β₃ as indicated by the line marked with open circles was assessedwith ¹²⁵I-fibrinogen. All data are expressed as the % of control bindingin the absence of inhibitor. Data points are the average of triplicatepoints in which the error was less than 11% of the total specificbinding. Non-specific binding was determined by competition with RGDpeptides and was normally less than 10% of the total bound counts.

FIGS. 4A, 4B, 4C and 4D illustrate that synthetic peptides of thisinvention derived from semi-synthetic antibodies inhibit integrinfunction and display the specificity of the whole antibody. Peptidesderived from the heavy CDR3 protein sequence of Fab 9, linear peptideGSFGRGDIRNG (SEQ ID NO 68) and cyclized peptide CSFGRGDIRNC (SEQ ID NO12), the latter of which was also prepared in linear form, weresynthesized as described in methods. Data is presented as described forFIG. 3.

In FIG. 4A, a linear peptide, designated Lin-9 having SEQ ID NO 68, wastested for the ability to block ¹²⁵I-vitronectin binding to α_(v)β₃(open triangles), α_(IIb)β₅ (closed circles) and ¹²⁵I-fibrinogen bindingto α_(IIb)β₃ (closed triangles). The peptide ranged in concentrationfrom 10⁻⁵ to 10⁻⁹ M.

In FIG. 4B, peptide GRGDSP (SEQ ID NO 66) was tested in an identicalassay in the same concentration range as above. All data are expressedas the % of control binding in the absence of inhibitor. Data areexpressed as the average of triplicate data points. This isrepresentative of four experiments in which nearly identical resultswere obtained in each repetition.

In FIG. 4C, peptides derived from HCDR3 of MTF-40, circular peptideCSFGRNDSRNC (SEQ ID NO 69) and linear peptide GCSFGRNDSRNCY (SEQ ID NO70) were also synthesized. The cyclic peptide data is indicated by opensquares and open triangles and linear peptide data is indicated byclosed circles and closed triangles forms. The ability of these peptidesto block ligand binding to α_(v)β₃ (closed triangles and open squares)and α_(IIb)β₃ (closed circles and open triangles) was compared. Thepeptides were tested at a concentration range of 10⁻⁵ to 10⁻¹¹ M. Alldata are expressed as the percent of control binding in the absence ofinhibitor. Data points are the average of triplicate values. Thisexperiment is representative of three repetitions in which nearlyidentical results were obtained.

In FIG. 4D, similar measurements were made for peptides derived fromHCDR3 of MTF-32, circular peptide CSFGRTDQRNC (SEQ ID NO 71) and linearpeptide GCSFGRTDQRNCY (SEQ ID NO 72). Linear (closed triangles andclosed circles) and cyclic (open triangles and open squares) peptideswere tested for the ability to block ligand binding to α_(v)β₃ (closedtriangles and open squares) and α_(IIb)β₃ (closed circles and opentriangles). Again the data are the average of triplicate points and theexperiment is representative of three repetitions with similar results.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 CFR §1.822(b)(2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left-to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase“amino acid residue” is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 CFR 1.822(b)(4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Recombinant DNA (rDNA) molecule: a DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature. rDNA's not having a common biological origin,i.e., evolutionarily different, are said to be “heterologous”.

Vector: A rDNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of genes encoding for one or morepolypeptides are referred to herein as “expression vectors”.Particularly important vectors allow cloning of cDNA (complementary DNA)that are produced from mRNAs produced using reverse transcriptase.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Antibody: The term antibody in its various grammatical forms is usedherein to refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and portions of an immunoglobulin molecule, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v).

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Althoughhistorically a monoclonal antibody was produced by immortalization of aclonally pure immunoglobulin secreting cell line, a monoclonally purepopulation of antibody molecules can also be prepared by the methods ofthe present invention.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptidesand a linking sequence to operatively link the two polypeptides into onecontinuous polypeptide. The two polypeptides linked in a fusionpolypeptide are typically derived from two independent sources, andtherefore a fusion polypeptide comprises two linked polypeptides notnormally found linked in nature.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′- to 3′-direction along theRNA transcript.

Cistron: A sequence of nucleotides in a DNA molecule coding for an aminoacid residue sequence and including upstream and downstream DNAexpression control elements.

Leader Polypeptide: A short length of amino acid sequence at the aminoend of a polypeptide, which carries or directs the polypeptide throughthe inner membrane and so ensures its eventual secretion into theperiplasmic space and perhaps beyond. The leader sequence peptide iscommonly removed before the polypeptide becomes active.

Reading Frame: A particular sequence of contiguous nucleotide triplets(codons) employed in translation. The reading frame depends on thelocation of the translation initiation codon.

B. Methods for Producing Binding Sites

The present invention relates generally to methods for producingpolypeptide-defined binding sites. The method involves the preparationof libraries of different binding sites on a phage display protein usingdegenerate oligonucleotides and primer extension reactions toincorporate the degeneracies into the binding site region of the displayprotein.

A binding site, described in more detail below, is any region of aprotein or polypeptide that participates in protein-target moleculeinteractions, and therefore the identification of the primary sequenceof a binding site is important in constructing a functional bindingsite-containing molecule.

The display of a binding site on a filamentous phage is essential to thepresent invention as it provides the ability to screen for capacity ofthe displayed binding site to bind to a preselected target molecule.Display is described in more detail but generally involves thepreparation of a fusion protein containing a membrane anchor of thefilamentous phage gene III or gene VIII protein fused to the polypeptideto be displayed that forms a binding site to be tested. Peptide displayvectors using hexapeptides have been generally described before byCwirla et al., Proc. Natl. Acad. Sci., USA, 87:6378-6382 (1990); Scottet al., Science, 249:386-390 (1990); and Devlin et al., Science,249:404-406 (1990).

According to the present methods, the use of an antibody heavy or lightchain as the display support structure on a recombinant filamentousphage, or phagemid, is particularly preferred. The use of an antibodysupport for a binding site provides a “scaffold” for presenting aconformationally constrained polypeptide to preselected bindingconditions. In this embodiment, a binding site is engineered into acomplementarity determining region (CDR) of an immunoglobulin heavy orlight chain that is presented on a phagemid, and the phagemid can bescreened for binding to a preselected target molecule.

The introduction of a binding site onto a display phagemid involves theuse of degenerate oligonucleotides to introduce into an immunoglobulinCDR region a series of different but related polypeptides defining afamily of binding sites. The degenerate oligonucleotides contain regionsof degeneracy to produce a library of different binding site structures,but also contain a region that is conserved that define a minimumbinding site polypeptide known to represent an important recognitionportion of the binding site to be developed. An example of a minimumbinding site polypeptide is the RGD tripeptide found in many ligandsthat bind to integrins.

Thus, the method comprises the basic step of introducing a nucleotidesequence that codes for an amino acid sequence that defines a bindingsite into a gene that codes a phagemid display protein. The introductionstep is conducted by amplifying a display region of the display proteingene by a primer extension reaction using a primer oligonucleotidehaving (1) regions complementary to the display protein gene, (2)regions that code a minimum binding site and (3) regions of degeneracythat introduce the variability into the resulting pool of displayproteins having binding sites.

After the primer extension reaction, which may be accomplished in avariety of modes including polymerase chain reaction (PCR), crossoverPCR, and the like, the resulting population of display protein genes areexpressed in phagemids to form a population of phagemid particles havingthe display proteins with binding sites on the particle surface. Thepopulation of particles are then screened for the presence of particlescontaining a binding site that binds to a preselected target molecule.

In one preferred embodiment, a library was prepared having the RGDbinding site introduced into a display protein comprised ofimmunoglobulin variable domain. In this approach, the domainsimmediately adjacent to the codons for “RGD” were randomized usingdegenerate oligonucleotides in a primer extension reaction.

By this approach, large populations of antibody molecules were obtainedwhich contain the RGD tripeptide in the binding site, and which bind toselected integrins against which they were screened for binding.Exemplary antibodies produced by this are described herein.

In a related embodiment, the invention provides a method for preparingadditional binding sites using a binding site produced by the abovemethods as a starting material, which can be further mutagenized andscreened for binding activity. By using a binding site having a desiredengineered binding specificity as a starting material, one can increasethe binding affinity or binding specificity, or both, to produceimproved binding sites. In particular, it may be desirable to preserveaspects of the binding site, referred to as motifs, and at the same timeselectively mutagenize other portions of the binding site. Thecombination of random mutagenesis of regions of the binding sitetogether with preservation of selected motifs is referred to as selectedbinding site motif engineering.

In a particularly preferred embodiment, selected binding site motifengineering involves first optimizing one motif, and then optimizing asecond motif. As an example, described in detail in the Examples, theRGD motif was first introduced into an antibody to produce a bindingsite specific for the integrins α_(IIb)β₃ and α_(v)β₃. Subsequently, apreferred antibody produced in the first step was sequenced, and asecond oligonucleotide was designed that preserves sites flanking theRGD motif, but intentionally randomizes the RGD domain, in order toidentify a motif in place of RGD that exhibits enhanced specificity.

Exemplary oligonucleotides useful for such selected motif engineeringare described herein.

C. Binding Site Polypeptides

A binding site can be any polypeptide sequence, typically about 3 to 20amino acid residues in length but can be from 3 up to 50 amino acidresidues in length, that defines a region of a protein or polypeptidewhich selectively interacts with another molecule or family of relatedmolecules, referred to as target molecules.

As is known in protein biochemistry, proteins, and therefore bindingsites as defined herein, can interact with a wide diversity of moleculesincluding other proteins, in the form of receptors, tissue structures,and soluble proteins, polypeptides including degraded proteins,polypeptide hormones and ligands, lipid, oligosaccharides andcarbohydrates, nucleic acids and inorganic molecules.

As shown herein and by other work in the field, the amino acid residuesequence of a binding site can tolerate some degree of variability andstill retain a degree of capacity to bind the target molecule.Furthermore, changes in the sequence can result in changes in thebinding specificity and in the binding constant between a preselectedtarget molecule and the binding site. Thus, the present method isideally suited to producing binding sites derived from a referencebinding site in which the binding constant is increased, making a morepotent binding reagent, or in which the specificity is altered orincreased, making a more selective binding reagent.

The “reference” binding site is any known, or yet to be described, aminoacid sequence having the ability to selectively bind a preselectedagent. These reference binding sites can be taken from any protein knownto be involved in protein-target interactions in which the amino acidreside sequence of the binding domain of the protein is known.

Exemplary reference binding sites are derived from the RGD-dependentintegrin ligands, namely fibronectin, fibrinogen, vitronectin, vonWillebrand factor and the like, from the envelope glycoprotein ofviruses such as HIV gp120, EBV gp350/220, reovirus hemagglutinin, andthe like, from cellular receptors such as CR2 or CD4, from proteinhormones such as thyroid stimulating hormone (TSH), insulin, transferrinand the like, from apolipoproteins such as ApoE and ApoAI, fromimmunoglobulin CDRs, and from major histocompatibility complex class Ior class II proteins.

In producing a binding site according to the present invention, theminimum binding or recognition domain of the binding site can beidentified to increase the degree of possible variant binding sitesbased on the reference binding site. A minimum recognition domain is theminimum sequence of amino acids required to confer binding specificityto an otherwise random polypeptide. Where a minimum recognition domainis not known, a polypeptide having known binding properties can be used.An exemplary and prototype minimum recognition domain is the RGDtripeptide required in polypeptides which bind integrins in anRGD-dependent manner.

1. RGD-dependent Binding Sites

A preferred binding site is an RGD tripeptide-containing binding site,and is exemplary of the general methods described herein. Ligands havingthe RGD motif as a minimum recognition domain are well known, a partiallist of which includes, with the corresponding integrin target inparenthesis, fibronectin (α₃β₁, α₅β₁, α_(v)β₁, α_(v)β₁, α_(IIb)β₃,α_(v)β₃ and α_(v)β_(b)) fibrinogen (α_(M)β₂ and α_(IIb)β₁) vonWillebrand factor (α_(IIb)β₃ and α_(v)β₃) vitronectin (α_(IIb)β₃,α_(v)β₃ and α_(v)β₅). See, for example D'Souza et al., Trends in Bioch.Sci., 16:246-250 (1991).

Particularly preferred RGD dependent binding sites identified by thepresent methods have an amino acid residue sequence shown below, and areshown to bind the human platelet glycoprotein gpIIb/IIIa (α_(IIb)β₃)when present in a phagemid display protein:

Designation (SEQ ID NO) Amino Acid Residue Sequence IIb/IIIa-1 1VGCGALRGDDPWCYYMDV IIb/IIIa-17 2 VGYGRLRGDXPWCYYMDV^(a) IIb/IIIa-19 3VGCGRLRGDDPWCYYMDV VnRβ₃-5 (Fab 9) 4 VGCSFGRGDIRNCYYMDV MTFIIb/IIIa-1 5VGCSFGRGDDRNFYYMDV MTFIIb/IIIa-5 4 VGCSFGRGDIRNCYYMDV ^(a)The amino acidresidue sequence at residue “X” has not been accurately determined.

Other preferred RGD-dependent binding sites identified by the presentmethods have an amino acid residue sequence shown below, and are shownto bind human vitronectin receptor (VnR) α_(v)β₃ when present in aphagemid display protein:

Designation (SEQ ID NO) Amino Acid Residue Sequence VnRβ₃-4 6VGCTQGRGDWRSCYYMDV VnRβ₃-5 (Fab 9) 4 VGCSFGRGDIRNCYYMDV VnRβ₃-7 7VGCTYGRGDTRNCYYMDV VnRβ₃-8 8 VGCPIPRGDWRECYYMDV VnRβ₃-10 9VGCTWGRGDERNCYYMDV MTFVnRβ₃-3 4 VGCSFGRGDIRNCYYMDV

Still other preferred RGD-dependent binding sites identified by thepresent methods have an amino acid residue sequence shown below, and areshown to bind human vitronectin receptor (VnR) α_(v)β₅ when present in aphagemid display protein:

Designation (SEQ ID NO) Amino Acid Residue Sequence VnRβ₅-6 10VGCDKRRGDRPRCYYMDV VnRβ₅-11 11 VGCSRRRGDRPQCYYMDV

The specificity of the resulting phagemid display proteins were shown inthe Examples to distinguish species of RGD-dependent receptors. That is,some selectively bound IIb/IIIa, some bound vitronectin receptorα_(v)β₃, and some bound vitronectin receptor α_(v)β₅, establishing thepower of the present methods to produce unique and specific bindingsites.

As described further herein, a preferred and exemplary phage displayprotein is an immunoglobulin heterodimer in which the fusion to aphagemid membrane anchor is through an immunoglobulin heavy chainpolypeptide. In addition, a human monoclonal antibody in the form of asoluble Fab fragment can readily be prepared from the phagemid displayvector. In this regard, the resulting human monoclonal antibody (Mab),whether a Mab, Fab and the like, produced having, for example, bindingsite IIb/IIIa-1 above is referred to generally as Mab IIb/IIIa-1 toconnote the presence of the IIb/IIIa-1 binding site.

A preferred embodiment is a peptide containing the amino acid residuesequence of the binding site derived from an immunoglobulin heterodimerhaving a preselected binding specificity, such as IIb/IIIa-1. Thecorresponding peptide is referred to generally as peptide IIb/IIIa-1 toconnote the presence of the IIb/IIIa-1 binding site. A preferred peptidehas the amino acid residue sequence CSFGRGDIRNC (SEQ ID NO 12) derivedfrom the binding site of VnRβ₃-5, also referred to as Fab 9. Peptides ofthis invention are contemplated for use in both linear and circularizedtopologies. As described in the Examples, the VnRB3-5-derived peptideinhibits the binding of the high affinity ligand, vitronectin, to thevitronectin receptor, α_(v)β₃, equivalent to that seen with the solubleFab from which the peptide is derived.

2. Non-RGD-dependent Integrin Binding Sites

In a further embodiment, a binding site that mimics a non-RGD-dependentbinding site on an integrin receptor and having the binding specificityof a high affinity ligand that recognizes the selected integrin iscontemplated. Preferred non-RGD-dependent binding sites identified bythe present methods as described in herein have an amino acid residuesequence shown below and are shown to bind human vitronectin receptor(VnR) α_(v)β₃ when present in a phagemid display protein.

Designation (SEQ ID NO) Amino Acid Residue Sequence MTFVβ₃-18 13VGCSFGRADTRNCYYMDV MTFVβ₃-1 14 VGCSGFRVDDRNCYYMDV MTFVβ₃-9 15VGCSFGRQDARNCYYMDV MTFVβ₃-15 16 VGCSFGRSDVRNCYYMDV MTFVβ₃-12 17VGCSFGRADRRNCYYMDV MTFVβ₃-19 18 VGCSFGRSDVRNFYYMDV

Still other preferred non-RGD-dependent binding sites identified by thepresent methods as described herein have an amino acid residue sequenceshown below, and are shown to bind human platelet glycoproteingpIIb/IIIa when present in a phagemid display protein:

Designation (SEQ ID NO) Amino Acid Residue Sequence MTFIIb-IIIa-8 19VGCSFGRTXTRNCYYMDV MTFIIb/IIIa-14 20 VGCSFGRQDVRNCYYMDV MTFIIb/IIIa-1221 VGCSFGRDDGRNCYYMDV MTFIIb/IIIa-13 22 VGCSFGRWDARNCYYMDVMTFIIb/IIIa-16 23 VGCSFGXGDRRNCYYMDV MTFIIb/IIIa-7 24 VGCSFGKRDMRNCYYMDVMTFIIb/IIIa-10 25 VGCSFGKGDNRNCYYMDV MTFIIb/IIIa-2 26 VGCSFGRTDQRNCYYMDVMTFIIb/IIIa-19 27 GVRRVLGNQGSFLPGWDV MTFIIb/IIIa-21 28VGCSFGRXDGRNFYYMDV MTFIIb/IIIa-23 29 VGCSFGRRDERNCYYMDV MTFIIb/IIIa-2530 VGCSFGRNDARNCYYMDV MTFIIb/IIIa-31 31 VGCSFGRRDERNCYYMDVMTFIIb/IIIa-32 32 VGCSFGRTDTRNCYYMDV MTFIIb/IIIa-33 33VGCSFGRADNRNCYYMDV MTFIIb/IIIa-40 34 VGCSFGRNDSRNCYYMDV

The amino acid residue sequence at residue “X” in SEQ ID NOs 19, 23, and28 have not been accurately determined.

3. HIV Gp120 Binding Site

In another embodiment, a binding site that mimics a binding site on theCD4 lymphocyte receptor and having binding specificity for HIV gp120 iscontemplated. The minimum recognition domain of a preferred gp120 sitehas the amino acid residue sequence GNQGSFL (SEQ ID NO 35).

4. EBV Gp350/220 Binding Site

In another embodiment, a binding site that mimics a binding site on thecell receptor CR2 and having binding specificity for the EBV gp350/220receptor is contemplated. The minimum recognition domain of a preferredEBV gp350/220 binding site has the amino acid residue sequence EDPGFFNE(SEQ ID NO 36) or EDPGKQLYNVE (SEQ ID NO 37).

5. Other Binding Sites

Numerous other binding sites are contemplated by the present invention,and are readily obtainable by the present screening methods. Preferredminimum recognition domains of binding sites for use in the inventionare described below.

The insulin receptor binding site on insulin has the amino acid residuesequence: RLFFNYLVIFEMVHLKE (SEQ ID NO 38).

The reovirus receptor binding site on the viral hemagglutinin proteinhas the sequence: IVSYSGSGLN (SEQ ID NO 39).

The fibrinogen receptor binding site on fibrinogen A alpha has thesequence: STSYDRGDS (SEQ ID NO 40).

Thyroid hormone receptor has two preferred binding sites on thyroidstimulating hormone (TSH), and TSH has two forms. The TSHα binding sitesequences are RSKKTML (SEQ ID NO 41) and ITSEAT (SEQ ID NO 42). The TSHβbinding site sequences are NGKLFL (SEQ ID NO 43) and FSVPVALS (SEQ ID NO44).

The LDL receptor binding site on the ApoE protein has the sequence:(LRX₁LRKRLLX₂)₂ (SEQ ID NO 45), where X₁ can be K or A, and where X₂canbe R or A.

The lipid A binding site has the sequence: IKTKKFLKKT (SEQ ID NO 46).

The lecithin-cholesterol acyltransferase (LCAT) binding site on theApoAI protein has the sequence: PYLLDFQKKWQEE (SEQ ID NO 47).

The Mac-1 integrin receptor binding site on fibrinogen D-30 fragment hasthe sequence: QKRLDGS (SEQ ID NO 48).

D. Phagemid Display Proteins

The display of the binding site on a phagemid can be accomplished on anyof the surface proteins of the filamentous phage particle, althoughparticularly preferred are display proteins comprising gene III or geneVIII protein, as described herein. The use of gene III or gene VIIIprotein as a display protein on filamentous phage has been extensivelydescribed elsewhere herein.

Particularly preferred display proteins are fusions involving the use ofthe phage particle membrane anchor derived from gene III or gene VIIIfused to an immunoglobulin heavy or light chain as described herein. Inthis embodiment, the binding site is displayed in a complementarydetermining region (CDR) of the immunoglobulin heavy or light chain,which in turn is a fusion to the membrane anchor domain of the phage'sgene III or gene VIII protein.

When using an immunoglobulin heavy or light chain as the displayprotein, it is preferred to position the binding site within one or morecomplementary determining regions, CDR1, CDR2 or CDR3. Using the Kabatimmunoglobulin amino acid residue sequence position numbering system,the light chain CDR's are as follows: CDR1 (residues 23-35), CDR2(residues 49-57), and CDR3 (residues 88-98); and the heavy chain CDRsare as follows: CDR1 (residues 30-36), CDR2 (residues 49-66), and CDR3(residues 94-103). See, Kabat et al., “Sequences of Proteins ofImmunological Interest”, Fourth ed., NIH, (1987).

When inserting a binding site into a CDR of an immunoglobulin fusiondisplay protein, some, most or all of the CDR domain can be removed andsubstituted by the inserted binding site. It has been learned that theCDR is very accommodating to variably sized inserts without disruptingthe ability of the immunoglobulin to assemble and display the insertedamino acid residue sequence.

In one embodiment, the phage display protein is engineered to includestabilization features in addition to the stabilization provided by thenative structure of the display protein. To that end, cysteine residuescan be coded for by the oligonucleotide, such that disulfide bridges canbe formed. The placement of the cysteine residues can be varied, suchthat a loop structure of from about 5 to 20 amino acid residues isformed. An exemplary construction with cysteines is shown in the bindingsites IIb/IIIa-1, IIb/IIIa-19, VnRβ₃ sites -4, -5, -7, -8 and -10, andVnRβ₅ sites -6 and -11.

In another embodiment, a phage display protein can be engineered tocontain multiple binding sites. For example, using the heavy chainimmunoglobulin as exemplary, binding sites can be introduced separatelyinto one or more of the CDRs designated CDR1, CDR2 and CDR3.Additionally, one can introduce binding sites into a heavy chain CDR anda light chain CDR, into multiple heavy and light chain CDRs, and thelike combinations.

E. Oligonucleotides

The preparation of a binding site according to the present inventioninvolves the use of synthetic oligonucleotides designed to introduce aputative binding site into a display protein. Furthermore, theoligonucleotide strategies described herein have particular advantagesin creating in a single reaction an extremely large population ofdifferent binding sites by the use of degenerate oligonucleotides.

1. Oligonucleotides Used to Engineer a Minimum Binding Site

The general structure of an oligonucleotide for use in one of thepresent methods has the general formula JOYOQ, where J and Q defineregions of homology to regions of the display protein gene which flankthe site in which a binding site is to be inserted, O defines region ofdegeneracy in which variable amino acid residues are introduced, and Ydefines a minimum recognition domain of the binding site that is beingintroduced into the display protein's binding site.

The number of nucleotides for regions O or Y can vary widely, but mustbe in triplets so as to preserve the reading frame of the displayprotein. Typically, J and Q are of sufficient length to conferhybridization specificity with the template during the primer extensionreaction. Thus, J and Q are typically each at least 6 nucleotides, andpreferably each at least 9 nucleotides in length, although they can be12, 15, 18, 21 and up to about 24 nucleotides in length. The O's aretypically of a widely variable length coding typically from 3 to 20amino acid residues. Y defines a minimum recognition domain of about 3to 40 amino acids residues in length, although, typically 3 to 10residues are preferred.

Where the display protein is an immunoglobulin, the homologies aredirected to the immunoglobulin framework regions (FR) that flank the CDRinto which the binding site is to be inserted.

Thus, in one embodiment, the invention contemplates an oligonucleotideuseful as a primer for producing a binding site in a polypeptide codedfor by a hybrid immunoglobulin heavy or light chain gene. Theoligonucleotide has 5′ and 3′ termini and comprises:

i) a nucleotide sequence of about 5 to 50 nucleotides in length at the3′ termini capable of hybridizing to a first (upstream) framework regionof the immunoglobulin gene;

ii) a nucleotide sequence of about 5 to 50 nucleotides in length at the5′ termini capable of hybridizing to a second (downstream) frameworkregion of the immunoglobulin gene; and

iii) a nucleotide sequence between said 5′ and 3′ termini according tothe formula:—X—[MNN]_(a)—Y—[MNN]_(b)—X—,where a and b are whole integers greater than zero, the sum of a and bis from 5 to 50, X is a trinucleotide encoding cysteine or a nativeamino acid residue coded by the immunoglobulin gene, N is independentlyany nucleotide, M is adenine (A) or cytosine (C) or analogs thereof, andY is a nucleotide sequence that encodes a minimum recognition domain ofsaid binding site, and wherein said 5′ and 3′ terminal nucleotidesequences have a length of about 6 to 50 nucleotides in length, andsequences complementary thereto.

Additionally contemplated for use in this invention is the complementarynucleotide sequence of an oligonucleotide formulation and identifiedsequence. Specifically, the complementary nucleotide for use in theinvention may have 5′ and 3′ termini between which is the nucleotideformulation —X—[NNK]_(a)—Y—[NNK]_(b)—X—. This alternative embodiment ofa complementary oligonucleotide thus hybridizes to the non-coding(antisense) strand of the template DNA.

Insofar as the use of a primer extension oligonucleotide (primer) inthis invention is for the purpose of introducing directed mutations intoa known immunoglobulin gene sequence, and insofar as one can mutagenizeeither the sense or antisense strand of a double stranded nucleic acidmolecule, the use of the term “encode” in its various expressions ismeant to refer to either the sense or antisense nucleotide sequence withthe understanding that it is the sense strand which is used to definethe corresponding amino acid residue sequence.

The choice of framework regions depends on the CDR into which thebinding site is to be inserted. Thus, for example, for an insertion intoCDR3, the 3′ and 5′ regions are selected as to be complementary innucleotide sequence to the coding strand defining FR3 and FR4 that flankCDR3, respectively, where the oligonucleotide is to be complementary tothe coding (sense) strand of the template DNA.

A preferred and exemplary CDR for insertion of a binding site is theCDR3 of immunoglobulin heavy chain. Particularly preferred is theimmunoglobulin heavy chain display protein present in the vectorspC3AP313 and p7EIII, described herein.

Oligonucleotides used in the present methods that are particularlypreferred for producing an RGD-dependent binding site have the formula:5′CTCCTCCTCCTCCTCGACGTCCATATAATAATT[MNN]_(a)ATCGCCACG[MNN]_(b)TGGCCCCACTCTCGCACAATAATA3′(SEQ ID NO 49), and preferably a is 3 and b is 3. An additionalpreferred oligonucleotide of this type has the formula: 5′CTCCTCCTCCTCCTCGACGTCCATATAATAGCA[MNN]_(a)ATCGCCACG[MNN]_(b)GCACCCCACTCTCGCACAATAATA3′(SEQ ID NO 50), and preferably a is 3 and b is 3. This latteroligonucleotide is designed to introduce cysteine residues into thedisplay protein flanking the binding site.

Oligonucleotides used in the present methods that are particularlypreferred for producing an HIV gp120 binding site have the formula: 5′CTCCTCCTCCTCCTCGACGTC[MNN]_(a)CAGAAAACTCCCTTGATTACC[MNN]_(b)ACCTCTCGCACAGTAATACACGGC3′(SEQ ID NO 51), and preferably a is 3 and b is 5.

Other oligonucleotides are described in the Examples.

2. Oligonucleotides Used to Create Motif Switching Engineered Sites

In another embodiment, the invention describes oligonucleotides for usein the present methods which are designed to introduce (switch) a newmotif into an engineered site. The oligonucleotide contains regions ofdegeneracy as before to produce a library of binding sites, except thatselected regions of the oligonucleotide are designed to preserve regionsof a preselected binding site.

For example, described herein, oligonucleotides were designed tointroduce a known binding site motif “RGD” into a CDR region of animmunoglobulin, with the redundancies flanking the RDG motif. In thepresent example, the oligonucleotide used is based on a known bindingsite directed to an RGD-dependent ligand. The oligonucleotide isdesigned such that known sequences adjacent to the RGD motif arepreserved, and the redundancy is introduced upon the RGD motif itself.The resulting binding sites do not necessarily contain the RGD motif,and in some cases a binding site is produced that does not contain RGD,but that does bind the target (receptor) with higher affinity.

A structure of an oligonucleotide for use in this motif switchingembodiment has the general formula JLOPQ, where J and Q define regionsof homology to regions of the display protein gene which flank the sitein which a binding site is to be inserted (e.g., immunoglobulinframework domains as before), O defines a region of degeneracy in whichvariable amino acid residues are introduced, and L and P define domainsof the binding site that is being preserved in the mutagenesisprocedure. For the case of RGD, the region “O” overlays the RGD motif,resulting in scrambling of the sequence at that position, whereas theregions “L” and “P” adjacent to the RGD region are conserved. Theinclusion of regions J and Q is optional.

Thus, in one embodiment, the invention describes an oligonucleotideuseful as a primer for producing a binding site while preserving a motifin a polypeptide coded for by a immunoglobulin heavy or light chaingene, wherein the oligonucleotide has 5′ and 3′ termini according to thegeneral formula: -LOP-. The oligonucleotide comprises:

i) a nucleotide sequence at said 3′ termini capable of hybridizing to afirst framework region of said immunoglobulin gene;

ii) a nucleotide sequence at said 5′ termini capable of hybridizing to asecond framework region of said immunoglobulin gene; and

iii) a nucleotide sequence between said 5′ and 3′ termini according tothe formula:-L-[MNN]₄—P—where L and P are each one to ten trinucleotides encoding preselectedCDR sequences, N is independently any nucleotide, M is adenine (A) orcytosine (C) or analogs thereof, and sequences complementary thereto.

In a preferred embodiment, L is 5 trinucleotides in length, and P is 8trinucleotides in length. More preferably, L and P correspond insequence to a heavy chain immunoglobulin variable region amino acidresidue sequence. In a particularly preferred embodiment, theoligonucleotide has the nucleotide sequence 5′CTCCTCCTCCTCCTCGACGTCCATATAATAGCAATTCCT[MNN]₄CCCAAACGAGCACCCCACTCTCGCACAATAATA3′ (SEQ ID NO 52).

Oligonucleotides for use in the present invention can be synthesized bya variety of chemistries as is well known. An excellent review is“Oligonucleotide Synthesis: A Practical Approach”, ed. M. J. Gait, JRLPress, New York, N.Y. (1990). Suitable synthetic methods include, forexample, the phosphotriester or phosphodiester methods see Narang etal., Meth. Enzymol., 68:90, (1979); U.S. Pat. No. 4,356,270; and Brownet al., Meth. Enzymol., 68:109, (1979). Purification of synthesizedoligonucleotides for use in primer extension and PCR reactions is wellknown. See, example Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons, New York, (1987). Exemplary synthesis isdescribed in the Examples.

F. Primer Extension Reactions

The term “polynucleotide” as used herein in reference to primers, probesand nucleic acid fragments or segments to be synthesized by primerextension is defined as a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than 3. Itsexact size will depend on many factors, which in turn depends on theultimate conditions of use.

The term “primer” as used herein refers to a polynucleotide whetherpurified from a nucleic acid restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase and thelike, and at a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency, but may alternatively be indouble stranded form. If double stranded, the primer is first treated toseparate it from its complementary strand before being used to prepareextension products. Preferably, the primer is a polydeoxyribonucleotide.The primer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the agents for polymerization. The exactlengths of the primers will depend on many factors, includingtemperature and the source of primer. For example, depending on thecomplexity of the target sequence, a polynucleotide primer typicallycontains 15 to 25 or more nucleotides, although it can contain fewernucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with template.

The primers used herein are selected to be “substantially” complementaryto the different strands of each specific sequence to be synthesized oramplified. This means that the primer must be sufficiently complementaryto non-randomly hybridize with its respective template strand.Therefore, the primer sequence may or may not reflect the exact sequenceof the template. For example, a non-complementary nucleotide fragmentcan be attached to the 5′ end of the primer, with the remainder of theprimer sequence being substantially complementary to the strand. Suchnon-complementary fragments typically code for an endonucleaserestriction site. Alternatively, non-complementary bases or longersequences can be interspersed into the primer, provided the primersequence has sufficient complementarily with the sequence of the strandto be synthesized or amplified to non-randomly hybridize therewith andthereby form an extension product under polynucleotide synthesizingconditions.

Primers of the present invention may also contain a DNA-dependent RNApolymerase promoter sequence or its complement. See for example, Krieget al., Nuc. Acids Res., 12:7057-70 (1984); Studier et al., J. Mol.Biol., 189:113-130 (1986); and Molecular Cloning: A Laboratory Manual,Second Edition, Sambrook et al., eds., Cold Spring Harbor, N.Y. (1989).

When a primer containing a DNA-dependent RNA polymerase promoter isused, the primer is hybridized to the polynucleotide strand to beamplified and the second polynucleotide strand of the DNA-dependent RNApolymerase promoter is completed using an inducing agent such as E. coliDNA polymerase I, or the Klenow fragment of E. coli DNA polymerase. Thestarting polynucleotide is amplified by alternating between theproduction of an RNA polynucleotide and DNA polynucleotide.

Primers may also contain a template sequence or replication initiationsite for a RNA-directed RNA polymerase. Typical RNA-directed RNApolymerase include the QB replicase described by Lizardi et al.,Biotechnology, 6:1197-1202 (1988). RNA-directed polymerases producelarge numbers of RNA strands from a small number of template RNA strandsthat contain a template sequence or replication initiation site. Thesepolymerases typically give a one million-fold amplification of thetemplate strand as has been described by Kramer et al., J. Mol. Biol.,89:719-736 (1974).

The choice of a primer's nucleotide sequence depends on factors such asthe distance on the nucleic acid from the region of the display proteingene into which a binding site is being introduced, its hybridizationsite on the nucleic acid relative to any second primer to be used, andthe like.

The PCR reaction is performed using any suitable method. Generally itoccurs in a buffered aqueous solution, i.e., a PCR buffer, preferably ata pH of 7-9, most preferably about 8. Preferably, a molar excess of theprimer is admixed to the buffer containing the template strand. A largemolar excess of about 104:1 of primer to template is preferred toimprove the efficiency of the process.

The PCR buffer also contains the deoxyribonucleotide triphosphates DATP,dCTP, dGTP, and dTTP and a polymerase, typically thermostable, all inadequate amounts for primer extension (polynucleotide synthesis)reaction. The resulting solution (PCR admixture) is heated to about 90degrees Celsius (90 C) to 100 C for about 1 to 10 minutes, preferablyfrom 1 to 4 minutes. After this heating period the solution is allowedto cool to 54 C, which is preferable for primer hybridization. Thesynthesis reaction may occur at from room temperature up to atemperature above which the polymerase (inducing agent) no longerfunctions efficiently. Thus, for example, if DNA polymerase is used asinducing agent, the temperature is generally no greater than about 40 C.An exemplary PCR buffer comprises the following: 50 millimolar (mM) KCl;10 mM Tris-HCl; pH 8.3; 1.5 mM MgCl₂; 0.001% (wt/vol) gelatin, 200micromolar (uM) dATP; 200 uM dTTP; 200 uM dCTP; 200 uM dGTP; and 2.5units Thermus aquaticus DNA polymerase I (U.S. Pat. No. 4,889,818) per100 microliters (ul) of buffer.

The inducing agent may be any compound or system which will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E. coliDNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, reverse transcriptase, andother enzymes, including heat-stable enzymes, which will facilitatecombination of the nucleotides in the proper manner to form the primerextension products which are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

The inducing agent also may be a compound or system which will functionto accomplish the synthesis of RNA primer extension products, includingenzymes. In preferred embodiments, the inducing agent may be aDNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. These polymerases produce acomplementary RNA polynucleotide. The high turn over rate of the RNApolymerase amplifies the starting polynucleotide as has been describedby Chamberlin et al., The Enzymes, ed. P. Boyer, PP. 87-108, AcademicPress, New York (1982). Another advantage of T7 RNA polymerase is thatmutations can be introduced into the polynucleotide synthesis byreplacing a portion of cDNA with one or more mutagenicoligodeoxynucleotides (polynucleotides) and transcribing thepartially-mismatched template directly as has been previously describedby Joyce et al., Nuc. Acids Res., 17:711-722 (1989). Amplificationsystems based on transcription have been described by Gingeras et al.,in PCR Protocols, A Guide to Methods and Applications, pp 245-252,Academic Press, Inc., San Diego, Calif. (1990).

If the inducing agent is a DNA-dependent RNA polymerase and thereforeincorporates ribonucleotide triphosphates, sufficient amounts of ATP,CTP, GTP and UTP are admixed to the primer extension reaction admixtureand the resulting solution is treated as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process, as is known for PCR.

PCR is typically carried out by thermocycling i.e., repeatedlyincreasing and decreasing the temperature of a PCR reaction admixturewithin a temperature range whose lower limit is about 10 C to about 40 Cand whose upper limit is about 90 C to about 100 C. The increasing anddecreasing can be continuous, but is preferably phasic with time periodsof relative temperature stability at each of temperatures favoringpolynucleotide synthesis, denaturation and hybridization.

PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including “PCR Technology: Principles and Applications for DNAAmplification”, H. Erlich, ed., Stockton Press, New York (1989); and“PCR Protocols: A Guide to Methods and Applications”, Innis et al.,eds., Academic Press, San Diego, Calif. (1990), the teachings of whichare hereby incorporated by reference.

Preferred PCR reactions using the oligonucleotides and methods of thisinvention are described in the Examples.

G. Phage Display Vectors

The methods of the present invention for preparing binding sites involvethe use of phage display vectors for their particular advantage ofproviding a means to screen a very large population of expressed displayproteins and thereby locate one or more specific clones that code for adesired binding reactivity.

The use of phage display vectors derives from the previously describeduse of combinatorial libraries of antibody molecules based on phagemids.The combinatorial library production and manipulation methods have beenextensively described in the literature, and will not be reviewed indetail herein, except for those feature required to make and use uniqueembodiments of the present invention. However, the methods generallyinvolve the use of a filamentous phage (phagemid) surface expressionvector system for cloning and expressing antibody species of thelibrary. Various phagemid cloning systems to produce combinatoriallibraries have been described by others. See, for example thepreparation of combinatorial antibody libraries on phagemids asdescribed by Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366(1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991);Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kanget al., Proc. Natl. Acad. Sci., USA, 88:11120-11123 (1991); Barbas etal., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992); and Gram et al.,Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992), the disclosures ofwhich are hereby incorporated by reference.

1. Phage Display Vector Structure

A preferred phagemid vector of the present invention is a recombinantDNA (rDNA) molecule containing a nucleotide sequence that codes for andis capable of expressing a fusion polypeptide containing, in thedirection of amino- to carboxy-terminus, (1) a prokaryotic secretionsignal domain, (2) a heterologous polypeptide defining an immunoglobulinheavy or light chain variable region, and (3) a filamentous phagemembrane anchor domain. The vector includes DNA expression controlsequences for expressing the fusion polypeptide, preferably prokaryoticcontrol sequences.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface.

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged aminoacid residues normally found at the cytoplasmic face of the membrane andextending away from the membrane.

In the phage f1, gene VIII coat protein's membrane spanning regioncomprises residue Trp-26 through Lys-40, and the cytoplasmic regioncomprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa et al.,J. Biol. Chem., 256:9951-9958, 1981). An exemplary membrane anchor wouldconsist of residues 26 to 40 of cpVIII. Thus, the amino acid residuesequence of a preferred membrane anchor domain is derived from the M13filamentous phage gene VIII coat protein (also designated cpVIII or CP8). Gene VIII coat protein is present on a mature filamentous phage overthe majority of the phage particle with typically about 2500 to 3000copies of the coat protein.

In addition, the amino acid residue sequence of another preferredmembrane anchor domain is derived from the M13 filamentous phage geneIII coat protein (also designated cpIII). Gene III coat protein ispresent on a mature filamentous phage at one end of the phage particlewith typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure of filamentous phageparticles, their coat proteins and particle assembly, see the reviews byRached et al., Microbiol. Rev., 50:401-427 (1986); and Model et al., in“The Bacteriophages: Vol. 2”, R. Calendar, ed. Plenum Publishing Co.,pp. 375-456 (1988).

The secretion signal is a leader peptide domain of a protein thattargets the protein to the periplasmic membrane of gram negativebacteria. A preferred secretion signal is a pelB secretion signal. Thepredicted amino acid residue sequences of the secretion signal domainfrom two pelB gene product variants from Erwinia carotova are describedin Lei et al., Nature, 331:543-546 (1988).

The leader sequence of the pelB protein has previously been used as asecretion signal for fusion proteins (Better et al., Science,240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci., USA,86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci., USA,87:8095-8099 (1990)). Amino acid residue sequences for other secretionsignal polypeptide domains from E. coli useful in this invention asdescribed in Oliver, Escherichia coli and Salmonella Typhimurium,Neidhard, F. C. (ed.), American Society for Microbiology, Washington,D.C., 1:56-69 (1987).

DNA expression control sequences comprise a set of DNA expressionsignals for expressing a structural gene product and include both 5′ and3′ elements, as is well known, operatively linked to the cistron suchthat the cistron is able to express a structural gene product. The 5′control sequences define a promoter for initiating transcription and aribosome binding site operatively linked at the 5′ terminus of theupstream translatable DNA sequence.

The 3′ control sequences define at least one termination (stop) codon inframe with and operatively linked to the heterologous fusionpolypeptide.

In preferred embodiments, the vector utilized includes a prokaryoticorigin of replication or replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra chromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such origins ofreplication are well known in the art. Preferred origins of replicationare those that are efficient in the host organism. A preferred host cellis E. coli. For use of a vector in E. coli, a preferred origin ofreplication is ColE1 found in pBR322 and a variety of other commonplasmids. Also preferred is the p15A origin of replication found onPACYC and its derivatives. The ColE1 and p15A replicon have beenextensively utilized in molecular biology, are available on a variety ofplasmids and are described at least by Sambrook et al., in “MolecularCloning: a Laboratory Manual”, 2nd edition, Cold Spring HarborLaboratory Press (1989).

The ColE1 and p15A replicons are particularly preferred for use in oneembodiment of the present invention where two “binary” plasmids areutilized because they each have the ability to direct the replication ofplasmid in E. coli while the other replicon is present in a secondplasmid in the same E. coli cell. In other words, ColE1 and p15A arenon-interfering replicons that allow the maintenance of two plasmids inthe same host (see, for example, Sambrook et al., supra, at pages1.3-1.4). This feature is particularly important when using binaryvectors because a single host cell permissive for phage replication mustsupport the independent and simultaneous replication of two separatevectors, for example when a first vector expresses a heavy chainpolypeptide and a second vector expresses a light chain polypeptide.

In addition, those embodiments that include a prokaryotic replicon canalso include a gene whose expression confers a selective advantage, suchas drug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin, tetracycline, neomycin/kanamycin or chloramphenicol. Vectorstypically also contain convenient restriction sites for insertion oftranslatable DNA sequences. Exemplary vectors are the plasmids pUC8,pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond,Calif.) and pPL and pKK223 available from Pharmacia, (Piscataway, N.J.).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. Preferred vectorsare those capable of autonomous replication and expression of structuralgene products present in the DNA segments to which they are operativelylinked. Vectors, therefore, preferably contain the replicons andselectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase“operatively linked” means the sequences or segments have beencovalently joined, preferably by conventional phosphodiester bonds, intoone strand of DNA, whether in single or double stranded form. The choiceof vector to which a transcription unit or a cassette of this inventionis operatively linked depends directly, as is well known in the art, onthe functional properties desired, e.g., vector replication and proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

In a preferred embodiment, a DNA expression vector is designed forconvenient manipulation in the form of a filamentous phage particleencapsulating a genome according to the teachings of the presentinvention. In this embodiment, a DNA expression vector further containsa nucleotide sequence that defines a filamentous phage origin ofreplication such that the vector, upon presentation of the appropriategenetic complementation, can replicate as a filamentous phage in singlestranded replicative form and be packaged into filamentous phageparticles. This feature provides the ability of the DNA expressionvector to be packaged into phage particles for subsequent segregation ofthe particle, and vector contained therein, away from other particlesthat comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phagegenome, as is well known, that defines sites for initiation ofreplication, termination of replication and packaging of the replicativeform produced by replication (see, for example, Rasched et al.,Microbiol. Rev., 50:401-427, 1986; and Horiuchi, J. Mol. Biol.,188:215-223, 1986). A preferred filamentous phage origin of replicationfor use in the present invention is an M13, f1 or fd phage origin ofreplication (Short et al., Nucl. Acids Res., 16:7583-7600, 1988).

Preferred DNA expression vectors for cloning and expressing a phagemiddisplay protein of this invention are the dicistronic plasmid expressionvectors pC3AP313 and p7EIII described herein.

It is to be understood that, due to the genetic code and its attendantredundancies, numerous polynucleotide sequences can be designed thatencode a contemplated heavy or light chain immunoglobulin variableregion amino acid residue sequence. Thus, the invention contemplatessuch alternate polynucleotide sequences incorporating the features ofthe redundancy of the genetic code.

Insofar as the expression vector for producing a human monoclonalantibody of this invention is carried in a host cell compatible withexpression of the antibody, the invention contemplates a host cellcontaining a vector or polynucleotide of this invention. A preferredhost cell is E. coli, as described herein.

Preferred expression vectors and plasmids that produce a phagemiddisplay protein of this invention were deposited pursuant to BudapestTreaty requirements with the American Type Culture Collection (ATCC),Rockville, Md., as described herein.

2. Use of Phagemid Display Vectors to Produce a Binding Site

The method for producing a binding site in a phagemid display proteingenerally involves (1) introducing a binding site into a phagemiddisplay protein vector by primer extension with an oligonucleotide asdescribed herein, to form a large population of display vectors eachcapable of expressing different putative binding sites displayed on aphagemid surface display protein, (2) expressing the display protein andbinding site on the surface of a filamentous phage particle, and (3)isolating the surface-expressed phage particle using affinity techniquessuch as panning of phage particles against a preselected targetmolecule, thereby isolating one or more species of phagemid containing adisplay protein containing a binding site that binds a preselectedtarget molecule.

As a further characterization of the present invention the nucleotideand corresponding amino acid residue sequence of the gene coding thebinding site is determined by nucleic acid sequencing. The primary aminoacid residue sequence information provides essential informationregarding the binding site's reactivity.

An exemplary preparation of a binding site in the CDR3 region of a heavychain of an immunoglobulin is described in the Examples. The isolationof a particular vector capable of expressing a binding site of interestinvolves the introduction of the dicistronic expression vector able toexpress the phagemid display protein into a host cell permissive forexpression of filamentous phage genes and the assembly of phageparticles. Typically, the host is E. coli. Thereafter, a helper phagegenome is introduced into the host cell containing the phagemidexpression vector to provide the genetic complementation necessary toallow phage particles to be assembled. The resulting host cell iscultured to allow the introduced phage genes and display protein genesto be expressed, and for phage particles to be assembled and shed fromthe host cell. The shed phage particles are then harvested (collected)from the host cell culture media and screened for desirable bindingproperties. Typically, the harvested particles are “panned” for bindingwith a preselected molecule. The strongly binding particles are thencollected, and individual species of particles are clonally isolated andfurther screened for binding to the target molecule. Phage which producea binding site of desired binding specificity are selected.

As described in the Examples, one can use the preferred phagemidexpression vectors as a starting material to introduce any of a varietyof modifications into the expressed antibody's CDR region(s). Any of thedesired antibody CDR sequences disclosed herein can be introduced bywell known methods, including, but not limited to, the PCR-basedmutagenesis methods, oligonucleotide synthesis and other recombinant DNAmethodologies.

H. Human Monoclonal Antibodies

The present invention describes, in one embodiment, human monoclonalantibodies which contain a binding site as described herein and whichbind specifically to a preselected target molecule. The invention alsodescribes cell lines which produce the antibodies, methods for producingthe cell lines, and methods for producing the human monoclonalantibodies.

Insofar as a display protein of this invention on a phagemid particleis, in preferred embodiments, a fusion protein between an immunoglobulinheavy or light chain and a filamentous phage membrane anchor, it is tobe understood that the display protein is, in effect, an engineeredimmunoglobulin heavy or light chain into which a binding site has beenintroduced. Furthermore, in many embodiments, the expression of thedisplay protein is prepared on the phagemid surface as a heterodimerformed between immunoglobulin heavy and light chain polypeptides, withone or the other being a fusion protein with the membrane anchor. Thus,where the heavy chain is used as the fusion protein, a display proteinin preferred embodiments comprises a Fab fragment having an anchoredheavy chain associated with a light chain.

The preparation of cell lines producing monoclonal antibodies of theinvention is described in great detail further herein, and can beaccomplished using the phagemid vector mutagenesis methods describedherein, and using routine screening techniques which permitdetermination of the elementary binding patterns of the monoclonalantibody of interest indicative that the binding site has been produced.Thus, if a human monoclonal antibody being tested binds to thepreselected target molecule, then the human monoclonal antibody beingtested and the human monoclonal antibody produced by the cell lines ofthe invention are considered equivalent.

It is also possible to determine, without undue experimentation, if ahuman monoclonal antibody has the same (i.e., equivalent) specificity asa human monoclonal antibody of this invention by ascertaining whetherthe former prevents the latter from binding to a preselected targetmolecule. If the human monoclonal antibody being tested competes withthe human monoclonal antibody of the invention, as shown by a decreasein binding by the human monoclonal antibody of the invention in standardcompetition assays for binding to the target molecule when present inthe solid phase, then it is likely that the two monoclonal antibodiesbind to the same, or a closely related, epitope.

Still another way to determine whether a human monoclonal antibody hasthe specificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with thetarget molecule with which it is normally reactive, and then add thehuman monoclonal antibody being tested to determine if the humanmonoclonal antibody being tested is inhibited in its ability to bind thetarget molecule. If the human monoclonal antibody being tested isinhibited then, in all likelihood, it has the same, or functionallyequivalent, epitopic specificity as the monoclonal antibody of theinvention.

An additional way to determine whether a human monoclonal antibody hasthe specificity of a human monoclonal antibody of the invention is todetermine the amino acid residue sequence of the CDR regions of theantibodies in question. Antibody molecules having identical, orfunctionally equivalent, amino acid residue sequences in their CDRregions have the same binding specificity. Methods for sequencingpolypeptides is well known in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin the antibody, and in part by the light chain variableregion amino acid residue sequence.

Particularly preferred is a human monoclonal antibody having the bindingspecificity of the monoclonal antibody produced by an E. colimicroorganism or produced by a plasmid vector that is deposited with theATCC, as described further herein, or that is derived from a depositedplasmid vector, as described further herein.

Use of the term “having the binding specificity of” indicates thatequivalent monoclonal antibodies exhibit the same or similarimmunoreaction (binding) properties and compete for binding to apreselected target molecule.

The term “conservative variation or substitution” as used herein denotesthe replacement of an amino acid residue by another, biologicallysimilar residue. Examples of conservative variations include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, and the like.

The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies having the substituted polypeptide also bind to thepreselected target molecule. Analogously, another preferred embodimentof the invention relates to polynucleotides which encode the above notedheavy and/or light chain polypeptides and to polynucleotide sequenceswhich are complementary to these polynucleotide sequences. Complementarypolynucleotide sequences include those sequences which hybridize to thepolynucleotide sequences of the invention under stringent hybridizationconditions.

Human monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies.

The invention contemplates, in one embodiment, a monoclonal antibody ofthis invention produced by the present methods.

In another preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from a humanmonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods. A preferred method of producing a soluble Fab fragment isdescribed herein.

1. RGD-dependent Human Monoclonal Antibodies

a. Anti-GPIIb/IIIA Human Monoclonal Antibodies

In one embodiment, the invention describes a class of human monoclonalantibodies which immunoreact with human platelets and the plateletglycoprotein gpIIb/IIIa. One set of antibodies were produced by thepresent methods in which a binding site in a CDR3 domain of animmunoglobulin heavy chain was designed to contain an RDG minimumrecognition domain, as described in the Examples. Another set ofpreferred antibodies containing an RGD-dependent binding site wereproduced by the present methods for motif switching an existing bindingsite.

The resulting population of display vectors containing an RGD tripeptidein the display protein obtained by either of the methods of thisinvention were screened first for gpIIb/IIIa binding activity in anELISA assay in which gpIIb/IIIa were in the solid phase to identify apopulation of display vectors which bound gpIIb/IIIa. Thereafter,selected members of the population of display vectors with gpIIb/IIIabinding activity were isolated, soluble Fab expression was engineeredinto each of the selected vectors as described in the Examples, and theresulting soluble Fab were expressed, and screened for binding or otherfunctional activities.

Anti-gpIIb/IIIa human monoclonal antibodies were identified which havethe desirable property of inhibiting gpIIb/IIIa function in plateletsand other fibrinogen-gpIIb/IIIa ligand-receptor complex-mediated events,including inhibiting platelet aggregation and inhibiting thrombusformation. Data presented in the Examples illustrates anti-gpIIb/IIIahuman monoclonal antibodies that are potent inhibitors of plateletaggregation at concentrations of about 1-100 nanomolar (nM). The humanmonoclonal antibody designated Mab IIb/IIIa-19 exhibited particularlyhigh platelet aggregation inhibition IC₅₀ concentrations (concentrationrequired for 50% inhibition of platelet aggregation). The humanmonoclonal antibody designated Fab 9 exhibited an affinity of 5×1⁻⁹ Mtowards gpIIb/IIIa under the binding conditions reported in theExamples.

A preferred anti-gpIIb/IIIa human monoclonal antibody of this inventionhas the binding specificity of a monoclonal antibody comprising a heavychain immunoglobulin variable region amino acid residue sequenceselected from the group of binding site sequences consisting ofIIb/IIIa-1, IIb/IIIa-17, IIb/IIIa-19, Fab 9, MTFIIb/IIIa-1,respectively, SEQ ID NOs 1-5, and MTFIIb/IIIa-5 (SEQ ID NO 4) andconservative substitutions thereof.

Particularly preferred human monoclonal antibodies are those having theimmunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) where the light chain is the light chainencoded by the plasmid vector pC3AP313 described herein, and referred toas light chain 313, or L313, and the heavy chain has one of the recitedbinding sites, and conservative substitutions thereof. The designationof a human monoclonal antibody with a colon, e.g., H:L313 is to connotea H:L pair formed by the heavy and light chain, respectively, in whichthe light chain is the preferred L313 light chain described herein.

b. Anti-vitronectin Receptor Antibodies

In one embodiment, the invention describes a class of human monoclonalantibodies which immunoreact with human vitronectin receptor (VnR) andcells that contain the receptor. The antibodies were produced by thepresent methods in which a binding site in a CDR3 domain of animmunoglobulin heavy chain was designed to contain an RDG minimumrecognition domain, as described in the Examples.

The resulting population of display vectors containing an RGD tripeptidein the display protein were screened first for VnR binding activity inan ELISA assay in which either α_(v)β₃ or α_(v)β₅ were in the solidphase to identify a population of display phagemid vectors which boundthe solid phase antigen. Thereafter, selected members of the populationof display vectors with VnR binding activity were isolated, soluble Fabexpression was engineered into each of the selected vectors as describedin the Examples, and the resulting soluble Fab were expressed, andscreened for binding or other functional activities.

Anti-VnR human monoclonal antibodies are thereby identified which havethe desirable property of inhibiting VnR-mediated cell adhesion and cellmotility, and tumor metastasis.

A preferred anti-VnR human monoclonal antibody of this invention has thebinding specificity for α_(v)β₃ or α_(v)β₅, and more preferably isspecific for one but not the other VnR species. The human monoclonalantibody designated Fab 9 is particularly preferred because it exhibitedan affinity of 1×1⁻¹⁰ M towards α_(v)β₃ under the binding conditionsreported in the Examples, and was substantially less immunoreactive withα_(v)β₅.

Preferred antibodies which preferentially bind to α_(v)β₃ includeantibodies that include a sequence shown in SEQ ID NOs 4, 6, 7, 8 or 9.Preferred antibodies which preferentially bind to α_(v)β₅ includeantibodies that include a sequence shown in SEQ ID NOs 10 or 11.

Particularly preferred human monoclonal antibodies are those having theimmunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) where the light chain is the light chainencoded by the plasmid vector pC3AP313 described herein, and referred toas light chain 313, or L313, and the heavy chain has one of the recitedbinding sites, and conservative substitutions thereof. Particularlypreferred are antibodies in which the heavy chain is derived from theplasmid pC3AP313.

Therefore, in preferred embodiments the antibody composition containsantibody molecules that bind the α_(v)β₃ vitronectin receptor (VnR), andthat contain the α_(v)β₃-4, α_(v)β₃-5 (Fab 9), α_(v)β₃-7, α_(v)β₃-8,α_(v)β₃-10 or MTFVnRB3-3 binding site. One of the preferred antibodymolecules, Fab 9, is unique in that it immunoreacts with both gpIIb/IIIaand α_(v)β₃ but does not immunoreact with α_(v)β₅ as described in theExamples. Particularly preferred are human monoclonal antibodies havingthe heavy and light chain coded by the vector pC3AP313, and wherein theheavy chain includes one of the above-recited binding sites in the CDR3domain.

In another preferred embodiment, the antibody composition containsantibody molecules that bind the α_(v)β₅ vitronectin receptor (VnR), andthat contain the α_(v)β₅-6 or α_(v)β₅-11 binding site. Particularlypreferred are human monoclonal antibodies having the heavy and lightchain coded by the vector pC3AP313, and wherein the heavy chain includesone of the above-recited binding sites in the CDR3 domain.

c. Anti-HIV GP120 Human Monoclonal Antibody

Another preferred human monoclonal antibody produced by the presentmethods is prepared using the CD4 minimum recognition site describedherein, and is screened and selected for binding to the V3 looppolypeptide of HIV gp120.

2. Non-RGD-Dependent Human Monoclonal Antibodies

a. Anti-GPIIb/IIIA Human Monoclonal Antibodies

In one embodiment, the invention describes a class of human monoclonalantibodies which immunoreact with human platelets and the plateletglycoprotein gpIIb/IIIa. One set of preferred antibodies thatimmunoreact with selected integrin receptors through an RGD-independentbinding site were produced by the present methods for motif switching anexisting binding site.

The resulting population of display vectors containing a newlyengineered motif in place of the existing RGDI (SEQ ID NO 53) obtainedby the motif switching method of this invention performed in anoriginally selected display protein were screened first for gpIIb/IIIabinding activity in an ELISA assay in which gpIIb/IIIa were in the solidphase to identify a population of display vectors which boundgpIIb/IIIa. Thereafter, selected members of the population of displayvectors with gpIIb/IIIa binding activity were isolated, soluble Fabexpression was engineered into each of the selected vectors as describedin the Examples, and the resulting soluble Fab were expressed, andscreened for binding or other functional activities.

Anti-gpIIb/IIIa human monoclonal antibodies were identified which havethe desirable property of inhibiting gpIIb/IIIa function in plateletsand other fibrinogen-gpIIb/IIIa ligand-receptor complex-mediated events,including inhibiting platelet aggregation and inhibiting thrombusformation. Data presented in the Examples illustrates anti-gpIIb/IIIahuman monoclonal antibodies that are potent inhibitors of fibrinogenbinding to the receptor at concentrations of about 1-100 nanomolar (nM).The human monoclonal antibody designated MTFIIb/IIIa-10 (MTF-10) alsoinhibited the binding of vitronectin to α_(v)β₃ but it required 1×10⁻⁷ Mto only partially inhibit the binding. The human monoclonal antibodydesignated MTFIIb/IIIa-40 exhibited an affinity of about 1-4×1⁻⁹ Mtowards gpIIb/IIIa, depending upon the assay conditions, andparticularly depending upon the cation (Ca²⁺ or Mn²⁺). Similarly, MTF-32exhibited an affinity of about 1-3×1⁻⁹ M towards gpIIb/IIIa, dependingupon the assay conditions, and particularly depending upon the cation.Antibody MTF-10 exhibited an affinity of about 7-8×1⁻⁹ M towardsgpIIb/IIIa, depending upon the assay conditions and cation.

Insofar as the MTF series antibodies were produced by the methods of thepresent invention using Fab 9 as a starting material, it is noted thatthe resulting antibodies do not contain the “RGD” tripeptide in theresulting CDR region, although Fab 9 did contain an RGD motif. Thus, inone embodiment, a preferred monoclonal antibody, in its various forms,has a complementarity determining region amino acid residue sequencethat does not contain the RGD sequence. The preparation of high affinityantibodies which lack the RGD sequence in the CDR is unexpected because(1) RGD is well known to be an important motif for ligand bindingspecificity to gpIIb/IIIa, (2) natural ligands of gpIIb/IIIa (e.g.,fibrinogen) contain RGD, (3) the starting antibody Fab 9 used to preparethe MTF series antibodies contained RGD in its CDR domain, and (4)RGD-containing ligands competed with both Fab 9 and the MTF seriesantibodies for binding to gpIIb/IIIa.

A preferred anti-gpIIb/IIIa human monoclonal antibody of this inventionhas the binding specificity of a monoclonal antibody comprising a heavychain immunoglobulin variable region amino acid residue sequenceselected from the group of binding site sequences consisting of SEQ IDNOs 19-34 corresponding to the designated motif switched antibodies, andconservative substitutions thereof.

One preferred embodiment of the invention contemplates a humanmonoclonal antibody capable of immunoreacting with human plateletglycoprotein gpIIb/IIIa and inhibiting platelet aggregation, where theantibody immunoreacts with gpIIb/IIIa with an affinity of at least1×10⁻⁹ molar (M), and has a CDR sequence that does not contain the RGDsequence. 10 Particularly preferred in this embodiment is ananti-gpIIb/IIIa human monoclonal antibody having the binding specificityof a monoclonal antibody that comprises a heavy chain immunoglobulinvariable region amino acid residue sequence that has the sequencecharacteristics of a sequence selected from the group of binding sitesequences consisting of SEQ ID NOs 25, 32 and 34, and conservativesubstitutions thereof.

By the phrase “at least 1×10⁻⁹ M” is meant that, when measured usingstandard immunoreaction association and dissociation rate measurementsto determine binding affinity, the recited antibody has the specifiedaffinity, and preferably a greater binding affinity, such as 5×10⁻⁹ M,or even more (1×10⁻¹⁰ M). Immunoaffinity can be determined by any of avariety of methods, including standard radio-immuno assay, Skatchardanalysis, surface plasmon resonance, and the like methods, some of whichare described in the Examples.

Particularly preferred human monoclonal antibodies are those having theimmunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) where the light chain is the light chainencoded by the plasmid vector pC3AP313 described herein, and referred toas light chain 313, or L313, and the heavy chain has one of the recitedbinding sites, and conservative substitutions thereof. The designationof a human monoclonal antibody with a colon, e.g., H:L313 is to connotea H:L pair formed by the heavy and light chain, respectively, in whichthe light chain is the preferred L313 light chain described herein.

b. Anti-vitronectin Receptor Antibodies

In one embodiment, the invention describes a class of human monoclonalantibodies which immunoreact with human vitronectin receptor (VnR) andcells that contain the receptor. The antibodies that immunoreact withselected integrin receptors through an RGD-independent binding site wereproduced by the present methods for motif switching an existing bindingsite.

The resulting population of display vectors containing a newlyengineered motif in place of the existing RGDI (SEQ ID NO 53) obtainedby the motif switching method of this invention performed in anoriginally selected display protein were screened first for VnR bindingactivity in an ELISA assay in which either α_(v)β₃ or α_(v)β₅ were inthe solid phase to identify a population of display phagemid vectorswhich bound the solid phase antigen. Thereafter, selected members of thepopulation of display vectors with VnR binding activity were isolated,soluble Fab expression was engineered into each of the selected vectorsas described in the Examples, and the resulting soluble Fab wereexpressed, and screened for binding or other functional activities.

Anti-VnR human monoclonal antibodies are thereby identified which havethe desirable property of inhibiting VnR-mediated cell adhesion and cellmotility, and tumor metastasis.

A preferred anti-VnR human monoclonal antibody of this invention has thebinding specificity for α_(v)β₃ or α_(v)β₅, and more preferably isspecific for one but not the other VnR species.

Particularly preferred human monoclonal antibodies are those having theimmunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) where the light chain is the light chainencoded by the plasmid vector pC3AP313 described herein, and referred toas light chain 313, or L313, and the heavy chain has one of the recitedbinding sites, and conservative substitutions thereof. Particularlypreferred are antibodies in which the heavy chain is derived from theplasmid pC3AP313.

Therefore, in preferred embodiments the antibody composition containsantibody molecules that bind the α_(v)β₃ vitronectin receptor (VnR), andthat contain the motif switched human monoclonal antibodies having theamino acid residues sequences listed in SEQ ID NOs 13-18 correspondingto the designations described herein. One of the preferred antibodymolecules, Fab 9, is unique in that it immunoreacts with both gpIIb/IIIaand α_(v)β₃ but does not immunoreact with α_(v)β₅ as described in theExamples. Particularly preferred are human monoclonal antibodies havingthe heavy and light chain coded by the vector pC3AP313, and wherein theheavy chain includes one of the above-recited binding sites in the CDR3domain.

In another preferred embodiment, the antibody composition containsantibody molecules that bind the α_(v)β₅ vitronectin receptor (VnR), andthat contain the α_(v)β₃-6 or α_(v)β₃-11 binding site. Particularlypreferred are human monoclonal antibodies having the heavy and lightchain coded by the vector pC3AP313, and wherein the heavy chain includesone of the above-recited binding sites in the CDR3 domain.

I. Peptides Derived from Human Monoclonal Antibodies

Following the present invention, polypeptides having a desired bindingactivity have been produced using the present methods, and can beutilized in the form of an isolated polypeptide.

A polypeptide of the present invention comprises no more than about 100amino acid residues, preferably no more than about 60 residues, morepreferably no more than about 30 residues, and includes an amino acidresidue sequence defining a binding site of the present invention. Apolypeptide has the ability to bind with the ligand to which it wasdeveloped using the present mutagenesis and screening methods. Apreferred polypeptide has the capacity to bind to the integrin α_(IIb)β₃or α_(v)β₃.

In one embodiment the invention contemplates a polypeptide that includesan amino acid residue sequence that defines a binding site sequencederived from a monoclonal antibody of this invention. Insofar as apolypeptide is derived from the sequence of a monoclonal antibody withthe ability to immunoreact with a preselected receptor, the polypeptidealso binds to the preselected receptor, and can thereby function as ananalog to the native ligand for the receptor.

In one embodiment, a polypeptide of this invention includes an aminoacid residue sequence having the sequence characteristics of a sequenceaccording to the formula: -CSFGRGDIRNC- (SEQ ID NO 12). Preferably, apolypeptide includes an amino acid residue sequence according to theformula: -VGCSFGRGDIRNCYYMDV- (SEQ ID NO 4) or -GSFGRGDIRNG- (SEQ ID NO68). These polypeptides were derived from antibody Fab 9, and have theability to inhibit vitronectin binding to purified vitronectin receptorα_(v)β₃. A particularly preferred polypeptide has a sequence selectedfrom the group consisting of CSFGRGDIRNC (SEQ ID NO 12),VGCSFGRGDIRNCYYMDV (SEQ ID No 4) and GSFGRGDIRNG (SEQ ID NO 68).

Thus, an exemplary polypeptide described herein was obtained from thebinding site of a monoclonal antibody immunoreactive with the integrinsα_(IIb)β₃ or α_(v)β₃.

In addition, polypeptides according to the formula -CSFGRNDSRNC- or-GCSFGRNDSRNCY-, shown in SEQ ID NOs 69 and 70, respectively, wereobtained from the binding site of antibody MTF-40 described herein.Polypeptides including these sequences bind gpIIb/IIIa, and thereforecan be used to inhibit platelet adhesion and/or fibrinogen binding togpIIb/IIIa, and the functions accompanying binding.

Furthermore, polypeptides according to the formula -CSFGRTDQRNC- or-GCSFGRTDQRNCY-, shown in SEQ ID NOs 71 and 72, respectively, wereobtained from the binding site of antibody MTF-32 described herein.Polypeptides including these sequences also bind gpIIb/IIIa, andtherefore can be used to inhibit platelet adhesion and/or fibrinogenbinding to gpIIb/IIIa, and the functions accompanying binding.

Although the amino acid residue sequences that flank the specifiedsequence may vary, in one embodiment, the preferred flanking sequencesare those residues defining an immunoglobulin variable domain.

Polypeptides that bind to the integrins α_(IIb)β₃ or α_(v)β₃ describedherein inhibit normal interactions between the integrin and their normalligands. Thus, a polypeptide functions as an analog to the known ligandsfor the recited integrins. The polypetides can inhibit ligand binding tointegrin both in vitro in standardized assay conditions and in vivo.

Methods to measure the inhibition of binding between ligand and integrinare conveniently carried out in vitro in a standardized ELISA binding.Exemplary assays are detailed in the Examples herein, and can be used todetect the presence of inhibition.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of the binding site of the monoclonalantibody from which it was derived, so long as it includes the requiredsequence and is able to inhibit the binding of ligand to integrin asdescribed. Therefore, a polypeptide of this invention can have thesequence characteristics of a recited polypeptide, i.e., toleratenon-essential amino acid substitutions or modifications, so long as thepolypeptide functions substantially the same, i.e., is substantiallyequivalent.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide exhibits the expressed bindingactivity. Therefore, a present polypeptide can be subject to variouschanges, substitutions, insertions, and deletions where such changesprovide for certain advantages in its use. In this regard, a polypeptideof this invention corresponds to, rather than is identical to, thesequence of the corresponding binding site where one or more changes aremade and it retains its binding ability as defined herein.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the binding ability asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

A polypeptide is free of homoserine lactone when there is no detectablehomoserine lactone present in the polypeptide when subjected toconventional amino acid analysis able to indicate the presence ofhomoserine lactone or other amino acids. Amino acid analysis methodssuitable to detect homoserine lactone are generally well known in theart.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of antibody from which it was derived, it istypically because one or more conservative or non-conservativesubstitutions have been made, usually no more than about 30 numberpercent, and preferably no more than number percent of the amino acidresidues are substituted.

“Substantially homologous” means that a particular subject sequence ormolecule, for example, varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which does notresult in an adverse functional dissimilarity between reference andsubject sequences. For purposes of the present invention, amino acidsequences having greater than 90 percent similarity, equivalentbiological activity, and equivalent expression characteristics areconsidered substantially homologous.

Amino acid sequences having greater than 40 percent similarity areconsidered substantially similar. For purposes of determining homologyor similarity, truncation or internal deletions of the referencesequence should be disregarded, as should subsequent modifications ofthe molecule, e.g., glycosylation. Sequences having lesser degrees ofhomology and comparable bioactivity are considered equivalents.

Additional residues may also be added at either terminus of apolypeptide of this invention for the purpose of providing a “linker” bywhich the polypeptides of this invention can be conveniently affixed toa label or solid matrix, or carrier.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formepitopes cross-reactive with the corresponding integrin. Typical aminoacid residues used for linking are tyrosine, cysteine, lysine, glutamicand aspartic acid, or the like. In addition, a subject polypeptide candiffer, unless otherwise specified, from the natural sequence of thecorresponding antibody, the sequence being modified by terminal-NH₂acylation, e.g., acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases may be present. In thisregard, polypeptide cyclization is also a useful terminal modification.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

A polypeptide of the present invention also referred to herein as asubject polypeptide, can be synthesized by any of the techniques thatare known to those skilled in the polypeptide art, including recombinantDNA techniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in J. M. Steward and J. D. Young, “Solid PhasePeptide Synthesis”, W.H. Freeman Co., San Francisco, 1969; M. Bodanszky,et al., “Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976 andJ. Neienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983 for solid phase peptide synthesis, and E.Schroder and K. Kubke, “The Peptides”, Vol. 1, Academic Press (NewYork), 1965 for classical solution synthesis, each of which isincorporated herein by reference. Appropriate protective groups usablein such synthesis are described in the above texts and in J. F. W.McOmie, “Protective Groups in organic Chemistry”, Plenum Press, NewYork, 1973, which is incorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

A polypeptide of this invention can be used in the therapeutic methodsof this invention, inter alia, to inhibit the activity of thecorresponding integrin where inhibition is desired. Particularlypreferred is the use of the integrin-binding polypeptides as inhibitorsof integrin function by blocking ligand binding.

A polypeptide can be prepared and utilized in both a linear or in acyclized form. The preparation of cyclized polypeptides is discussed inthe Examples.

J. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with at least one species of human monoclonalantibody or polypeptide derived therefrom as described herein, dissolvedor dispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to ahuman patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified. Thus, an antibody molecule-containing composition can takethe form of solutions, suspensions, tablets, capsules, sustained releaseformulations or powders, or other compositional forms.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol, and other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water.

Exemplary of such additional liquid phases are glycerin, vegetable oilssuch as cottonseed oil, organic esters such as ethyl oleate, andwater-oil emulsions.

A therapeutic composition contains a human monoclonal antibody of thepresent invention, typically in an amount of at least 0.1 weight percentof antibody per weight of total therapeutic composition. A weightpercent is a ratio by weight of antibody to total composition. Thus, forexample, 0.1 weight percent is 0.1 grams of antibody per 100 grams oftotal composition.

Preferably, an antibody-containing therapeutic composition typicallycontains about 10 microgram (ug) per milliliter (ml) to about 100milligrams (mg) per ml of antibody as active ingredient per volume ofcomposition, and more preferably contains about 1 mg/ml to about 10mg/ml (i.e., about 0.1 to 1 weight percent).

A therapeutic composition in another embodiment contains a polypeptideof the present invention, typically in an amount of at least 0.1 weightpercent of polyeptide per weight of total therapeutic composition. Aweight percent is a ratio by weight of polyeptide to total composition.Thus, for example, 0.1 weight percent is 0.1 grams of polyeptide per 100grams of total composition.

Preferably, an polypeptide-containing therapeutic composition typicallycontains about 10 microgram (ug) per milliliter (ml) to about 100milligrams (mg) per ml of polypeptide as active ingredient per volume ofcomposition, and more preferably contains about 1 mg/ml to about 10mg/ml (i.e., about 0.1 to 1 weight percent).

K. Therapeutic Methods

In view of the benefit of using human monoclonal antibodies in vivo inhuman patients, the presently described antibodies are particularly wellsuited for in vivo use as a therapeutic reagent for blocking orinhibiting the function of the target molecule which the antibody binds.The peptides derived from the monoclonal antibodies described herein arealso contemplated for use in the therapeutic methods of this invention.The method comprises contacting a sample believed to contain the targetmolecule with a composition comprising a therapeutically effectiveamount of a human monoclonal antibody or peptide of this invention whichbinds the target molecule.

For in viva modalities, the method comprises administering to thepatient a therapeutically effective amount of a physiologicallytolerable composition containing a human monoclonal antibody or peptideof the invention.

The dosage ranges for the administration of the monoclonal antibodiesand peptides of the invention are those large enough to produce thedesired effect in which the disease symptoms mediated by the targetmolecule are ameliorated. The dosage should not be so large as to causeadverse side effects, such as hyperviscosity syndromes, pulmonary edema,congestive heart failure, and the like. Generally, the dosage will varywith the age, condition, sex and extent of the disease in the patientand can be determined by one of skill in the art.

The dosage can be adjusted by the individual physician in the event ofany complication.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (ug) per milliliter (ml) toabout 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, andusually about 5 ug/ml. Stated differently, the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or several days.

A therapeutically effective amount of a polypeptide of this invention istypically an amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (ug) per milliliter (ml) toabout 100 ug/ml, preferably from about 1 ug/ml to about 10 ug/ml. Stateddifferently, the dosage can vary from about 0.1 mg/kg to about 300mg/kg, and preferably from about 0.2 mg/kg to about 200 mg/kg, in one ormore dose administrations daily, for one or several days.

The human monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the target molecule can typically be accessed in the body bysystemic administration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverymeans are contemplated where there is a likelihood that the tissuetargeted contains the target molecule. Thus, human monoclonal antibodiesor polypeptides of the invention can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a human monoclonal antibody or apolyeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

1. Methods for Inhibiting Platelet GpIIb/IIIa Function

An anti-gpIIb/IIIA human monoclonal antibody or peptide derivedtherefrom containing a gpIIB/IIIa-binding site can be used to in vivo orin vitro modulate the function of gpIIb/IIIa on platelets. For instance,the human monoclonal antibody or peptide can be used in apharmaceutically acceptable composition that, when administered to ahuman subject in an effective amount, is capable of inhibiting theaggregation of platelets, and thereby decreasing the rate of thrombusformation. Thus, in vivo administration of an anti-gpIIb/IIIa humanmonoclonal antibody that inhibits platelet aggregation can be used invivo to modulate any physiological response initiated by plateletadhesion, such as coagulation and some inflammatory responses.

When this method is carried out in vivo, an effective amount of anantibody or peptide composition containing a physiologically tolerablediluent and antibody molecules that immunoreact with gpIIb/IIIa and thatinhibit platelet aggregation is intravenously administered to a mammal,and the mammal is maintained for a sufficient time period to allow theantibody molecules to immunoreact with any gpIIb/IIIa present and forman immunoreaction product and to allow the binding site containing thepeptide to bind to gpIIb/IIIa and form a peptide-receptor complex suchthat the normal ligand can no longer bind to the receptor.

In preferred embodiments the antibody composition contains antibodymolecules having a heavy chain that includes a binding site selectedfrom the group consisting of SEQ ID NOs 1-5, and 19-34 and having thelight chain L313. Particularly preferred are the monoclonal antibodieswhen expressed in the vector pC3AP313. In preferred embodiments, thepeptide composition for inhibiting platelet aggregation includes apolypeptide sequence selected from the group consisting of SEQ ID NOs12, 69, 70, 71 and 72, and conservative substitutions thereof.

2. Methods for Inhibiting HIV Gp120-mediated Events

The present invention describes in one embodiment a method for providingpassive immunotherapy to HIV disease in a human comprising administeringto the human an immunotherapeutically effective amount of an anti-HIVgp120 envelope glycoprotein monoclonal antibody of this invention.

A representative patient for practicing the present passiveimmunotherapeutic methods is any human exhibiting symptoms ofHIV-induced disease, including AIDS or related conditions believed to becaused by HIV infection, and humans at risk of HIV infection. Patientsat risk of infection by HIV include babies of HIV-infected pregnantmothers, recipients of transfusions known to contain HIV, users of HIVcontaminated needles, individuals who have participated in high risksexual activities with known HIV-infected individuals, and the like risksituations.

In one embodiment, the passive immunization method comprisesadministering a composition comprising more than one species of anti-HIVgp120 human monoclonal antibody of this invention, preferably directedto non-competing epitopes or directed to distinct serotypes or strainsof HIV, as to afford increased effectiveness of the passiveimmunotherapy.

A therapeutically (immunotherapeutically) effective amount of a humanmonoclonal antibody is a predetermined amount calculated to achieve thedesired effect, i.e., to neutralize the HIV present in the sample or inthe patient, and thereby decrease the amount of detectable HIV in thesample or patient. In the case of in vivo therapies, an effective amountcan be measured by improvements in one or more symptoms associated withHIV-induced disease occurring in the patient, or by serologicaldecreases in HIV antigens.

In preferred embodiments the antibody composition contains antibodymolecules expressed by the plasmid pC3AP313.

3. Methods for Inhibiting Vitronectin Receptor-mediated Events

An anti-vitronectin receptor human monoclonal antibody of this inventioncan be used to in vivo or in vitro to modulate the function ofvitronectin receptor present on a variety of cells.

The receptor for vitronectin (VnR) has been identified to be any one ofthe integrin heterodimers referred to as α_(v)β₃ or α_(v)β₅. Celladhesion to vitronectin is therefore mediated by VnR, and it has beendetermined that VnR-mediated adhesion is particularly important in tumorcell adhesion, migration and tumor metastases. See, for example,Leavesly et al., J. Cell Biol., 117:1101-1107 (1992). Thus, inhibitionof vitronectin interaction with VnR will perturb cell attachment eventsassociated with vitronectin, and will thereby block VnR-mediated eventssuch as tumor cell adhesion and migration.

Therefore, the invention also contemplates the use of an anti-VnR humanmonoclonal antibody or peptide-derived therefrom containing a VnRbinding site in a pharmaceutically acceptable composition that, whenadministered to a human subject in an effective amount, is capable ofinhibiting VnR binding to vitronectin, and thereby decreasing malignantcell adhesion and c ell migration. Thus, in vivo administration of ananti-VnR human monoclonal antibody or a VnR-specific peptide thatinhibits metastatic cell adhesion and migration can be used in vivo tomodulate any physiological response mediated by vitronectin binding toVnR, such as tumor cell attachment, and metastases.

When this method is carried out in vivo, an effective amount of anantibody or peptide composition respectively containing aphysiologically tolerable diluent and antibody or peptide molecules thatimmunoreact with VnR and that inhibit tumor cell migration isintravenously administered to a mammal, and the mammal is maintained fora sufficient time period to allow the antibody molecules or peptides toimmunoreact with any VnR present and form an immunoreaction product or apeptide/receptor complex.

In preferred embodiments the antibody composition contains antibodymolecules that bind the α_(v)β₃ vitronectin receptor (VnR) containingthe binding sites described herein and in SEQ ID NOs 4, 6, 7, 8, 9, and13-18. Particularly preferred are human monoclonal antibodies having theheavy and light chain coded by the vector pC3AP313, and wherein theheavy chain includes one of the above-recited binding sites in the CDR3domain.

In a related preferred embodiment, the antibody composition containsantibody molecules that bind the α_(v)β₅ vitronectin receptor (VnR), andthat contain the binding sites listed in SEQ ID NOs 10 and 11.Particularly preferred are human monoclonal antibodies having the heavyand light chain coded by the vector pC3AP313, and wherein the heavychain includes one of the above-recited binding sites in the CDR3domain.

In other preferred embodiments, a polypeptide composition for inhibitingvitronectin binding to VnR includes a polypeptide having a sequencecharacteristic of a sequence selected from the group consisting of SEQID NOs 4, 12, and 68, and conservative substitutions thereof.

EXAMPLES

The following examples relating to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

1. Preparation of Synthetic Binding Sites Within the Heavy Chain CDR3Domain of a Phagemid Fab Display Protein Produced by a DicistronicExpression Vector

A. Preparation of Nucleotide Sequences for Encoding Synthetic Bindingsites Containing the Peptide, Arginine-Glycine-Aspartic Acid (RGD)

The immunoglobulin gene phagemid expression vector, pMT12, containingthe heavy and light chain sequences for expressing the soluble form ofan antibody was used to prepare the synthetic binding site proteinscontaining the peptides, arginine-glycine-aspartic acid (RGD), of thisinvention as described below in Example 1A1). The phagemid Fab displayof an RGD peptide is one of the preferred binding sites prepared by themethods of this invention. The term “binding site” as defined herein isany region of a protein or polypeptide that participates inprotein-target molecule interactions. In the context of an RGD bindingsite, integrin receptors which bind RGD are contemplated as targetmolecules. For a review of RGD-dependent integrin receptors, seeGinsberg et al., TIBS, 16:246-250 (1991).

The RGD sequence has been shown to occur in 4 known structures, shakevenom “disintegrins” kistrin, echistatin, tenascin and the foot andmouth disease virus. In each of these structures, RGD is found in anextended flexible loop at the apex of the turn. The simple RGD motif isinsufficient for high affinity binding to RGD-specific receptors such asα_(v)/β₃ and gpIIb/IIIa. Binding is dependent on the correctconformational display of the motif as evidenced by perturbations inaffinity of peptides as a function of flanking sequences andconformational constraint conferred by cyclization. Simple placement ofthe RGD motif within a complementary determining region (CDR) of avariable region of an antibody thus would be insufficient to conferbinding with a useful affinity and specificity. For example, a naturalantibody containing a RGD motif at the C-terminal portion of the heavychain CDR3 failed to bind the vitronectin or gpIIb/IIIa integrinreceptors. Therefore, this invention has provided for the methods tooligonucleotides that result in the positioning of a RGD motif at theapex of an extended loop in the heavy chain CDR3 with flanking regionsthat conformationally optimize the display of the motif.

The binding sites of this invention as described herein thus wereincorporated into a CDR in the antibody heavy chain of pMT12. Thepreparation and characterization of the pMT12 phagemid expression vectorcontaining heavy and light chain sequences for the expression of humanmonoclonal antibodies has been described by Burton et al., Proc. Natl.Acad. Sci., USA, 88:10134-10137 (1991), the disclosure of which ishereby incorporated by reference.

The E. coli bacterial cells containing pMT12 soluble Fab-producingphagemids were deposited on Sep. 30, 1992, with the American TypeCulture Collection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC). Thedeposit of the plasmid-containing cells is listed under the name MT12and has been assigned the ATCC designation number 69079. This depositwas made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty).

The phagemid pMT12 was used in polymerase chain reaction (PCR)amplifications to create unique binding sites within the CDR3 of the Fabheavy chain in the phagemid. This human antibody was chosen as thepresenting scaffold as it was produced efficiently in E. coli and had anextended heavy chain CDR3 consisting of 18 instead of 16 amino acidresidues. Residues at the amino and carboxy terminal portion of theheavy chain CDR3 were maintained so as to retain the stem of theextended hair-pin loop. The RGD motif was introduced at Kabat positions100a-c ensuring its placement hear the apex of the turn.

For the PCR amplification, the 5′ end of the heavy chain beginning atframework 1 and extending to the 3′ end of CDR3 was amplified with twooligonucleotide primers. The position of the heavy chain CDR3 of pMT12corresponds to Kabat numbers 94-103. For preparing a syntheticRGD-containing binding site of this invention, a pool of oligonucleotideprimers shown below were synthesized with the complementary sequence forencoding the RGD peptide bordered by a degenerate region ((NNK)₃ where Kis G or T and N is A, C, G or T) that encoded 3 amino acids on bothsides of the RGD sequence. This region was further bordered by thenucleotide sequence for encoding the 5′ and 3′ CDR3 amino acid residuespresent in the pMT12 expression vector. Also contemplated for use in themethods of this invention for incorporating the RGD binding site intothe CDR3 is an oligonucleotide having the complementary sequence to theRGD primer described below. One having ordinary skill in the art candesign complementary oligonucleotides that will allow for theamplification of the same RGD-containing CDR3 in pMT12 as describedbelow.

Amplification products resulting from the procedure as described belowhad sequences for encoding an RGD peptide bordered by sequences forrandomly encoding 3 amino acids in the CDR3 region. The sequence NNKrepresents the coding strand sequence having the complementary sequenceNNM in the primer as read from the 3′ to 5′ direction. Thus, in theprimer as listed below the noncoding strand sequence is MNN as read inthe 5′ to 3′ direction. The coding triplet sequence NNK was designed toprevent the production of deleterious stop codons. The only stop codonthat could result from the expression of NNK would be an amber mutationthat is suppressed when the phagemid is expressed an amber-suppressinghost cell.

The PCR reaction resulted in the amplification of the region of theheavy chain fragment in the pMT12 phagemid vector clone beginning atframework region 1 (FR1) and extending to the end of the CDR3 domainwhich is approximately 450 base pairs (bp) in length. To amplify thisregion, the following primer pairs were used. The 5′ oligonucleotideprimer, FTX3, having the nucleotide sequence 5′ GCAATTAACCCTCACTAAAGGG3′(SEQ ID NO 54), hybridized to the noncoding strand of the heavy chaincorresponding to the region 5′ of and including the beginning of FR1.The 3′ oligonucleotide primer, RGD, having the nucleotide sequence 5′CTCCTCCTCCTCCTCGACGTCCCATATAATAATTMNNMNNMNNATCGCCACGMNNMNNMNNTGGCCCCACTCTCGCACAATAATA3′ (SEQ ID NO 49) where M is A or C andN is A, C, G or T, hybridized to the coding strand of the heavy chaincorresponding to CDR3. The oligonucleotide primers were synthesized byoperon Technologies, Alameda, Calif. The expected amino acid residuesequences in the CDR3 and flanking framework regions resulting from thePCR amplification with the 3′ primer above have the formula:-NH₂-YYCARVGPXXXRGDXXXNYYMDVEEEEE-COOH (SEQ ID NO 55), where X is Xaa,any amino acid. Refer to the Table of Correspondence and the SequenceListing for the identification of the amino acid residues presented insingle letter format.

The PCR reaction was performed in a 100 microliter (ul) reactioncontaining one microgram (ug) of each of oligonucleotide primers FTX3and RGD, 8 ul 2.5 millimolar (mM) dNTP's (dATP, dCTP, dGTP, dTTP), 1 ulTAQ POLYMERASE (Perkin-Elmer Corp., Norwalk, Conn.) 10 nanograms (ng) oftemplate pMT12, and 10 ul of 10× PCR buffer purchased commercially(Perkin-Elmer Corp.). Thirty-five rounds of PCR amplification in aPerkin-Elmer Cetus 9600 GeneAmp PCR System thermocycler were thenperformed. The amplification cycle consisted of denaturing at 94 degreesC. (94 C) for one minute, annealing at 47 C for one minute, followed byextension at 72 C for two minutes. To obtain sufficient quantities ofamplification product, 15 identical PCR reactions were performed.

The resultant PCR amplification products containing the sequence forencoding an RGD peptide were then gel purified on a 1.5% agarose gelusing standard electroelution techniques as described in “MolecularCloning: A Laboratory Manual”, Sambrook et al., eds., Cold SpringHarbor, N.Y. (1989). Briefly, after gel electrophoresis of the digestedPCR amplified Fab-display encoding synthetic binding sites, the regionof the gel containing the DNA fragments of predetermined size wasexcised, electroeluted into a dialysis membrane, ethanol precipitatedand resuspended in buffer containing 10 millimolar (mM) Tris-HCl[Tris(hydroxymethyl)aminomethane-hydrochloride] at pH 7.5 and 1 mM EDTA(ethylenediaminetetraacetic acid) to a final concentration of 50nanograms/milliliter (ng/ml).

The purified RGD-expressing PCR products were then digested with therestriction enzymes XhoI and AatII to form a heavy chain antibodyfragment for directional ligation into a similarly digested phagemidexpression vector, designated pC3AP313, for the surface display of theexpressed antibodies containing the newly created synthetic bindingsites. The surface display phagemid expression vector, pC3AP313, hasbeen deposited with ATCC on Feb. 2, 1993 for use in this invention. Thedeposited vector has been assigned the ATCC accession number 75408. ThePC3AP313 expression vector containing the coat protein encoded bybacteriophage gene 3 was prepared as described in Example 2. Theligation procedure in creating expression vector libraries and thesubsequent expression of the synthetic binding site-containingantibodies is performed as described in Example 2.

B. Preparation of Nucleotide Sequences Encoding Cyclized SyntheticBinding Sites Containing the Peptide, Arginine-Glycine-Aspartic Acid(RGD)

For preparing a cyclized version of the synthetic RGD-containing bindingsite within CDR3 of a human Fab antibody of this invention, a pool ofoligonucleotide primers shown below were synthesized with the sequenceencoding the RGD peptide bordered by a coding strand degenerate region((NNK)₃ where K is G or T and N is A, C, G or T) for encoding 3 aminoacids on both sides of the RGD sequence. This region was furtherbordered by the nucleotide sequence for encoding the 5′ and 3′ CDR3amino acid residues in the pMT12 expression vector. The resultingamplification products had sequences for encoding an RGD peptidebordered by sequences for randomly encoding 3 amino acids in the CDR3region further bordered by sequences for encoding cysteine residues toprovide for an intra-loop disulfide linkage around the RGD syntheticbinding site. This thereafter enabled the sequences to be directlysynthesized using standard peptide methodology and cyclized to providefor the conformationally correct peptide.

The PCR reaction resulted in the amplification of the region of theheavy chain fragment in the pMT12 phagemid vector clone beginning atframework region 1 (FR1) and extending to the end of the CDR3 domainwhich is approximately 450 base pairs (bp) in length. To amplify thisregion, the following primer pairs were used. The 5′ oligonucleotideprimer, FTX3, having the nucleotide sequence 5′ GCAATTAACCCTCACTAAAGGG3′(SEQ ID NO 54), hybridized to the noncoding strand of the heavy chaincorresponding to the region 5′ of and including the beginning of FR1.The 3′ oligonucleotide primer, RGDC2, having the nucleotide sequence 5′CTCCTCCTCCTCCTCGACGTCCATATAATAGCAMNNMNNMNNATCGCCACGMNNMNNMNNGCACCCCACTCTCGCACAATAATA3′ (SEQ ID NO 50) where M is A or C andN is A, C, G or T, hybridized to the coding strand of the heavy chaincorresponding to CDR3. The oligonucleotide primers were synthesized byOperon Technologies, Alameda, Calif. The expected amino acid residuesequences in CDR3 and flanking framework regions resulting from the PCRamplification with the 3′ primer have the formula:-NH₂-YYCARVGCXXXRGDXXXCYYMDVEEEEE-COOH (SEQ ID NO 56), where X is Xaa,any amino acid. Refer to the Table of Correspondence and the SequenceListing for the identification of the amino acid residues presented insingle letter format.

The PCR reaction was performed as described in Example 1A. The resultantPCR products were purified, digested with XhoI and AatII and insertedinto the similarly digested pC3AP313 surface display expression vectorfor preparation of an expression library as described in Example 2.

C. Preparation of Nucleotide Sequences Encoding Synthetic Binding SitesContaining the Binding Site on CD4 that is Specific for the HIVGlycoprotein gp120

The binding site of the HIV glycoprotein, gp120, on CD4 has beensuggested to reside within the amino acid residues of 37 to 49 on CD4.These residues were amplified into CDR3 of the heavy chain in thepC3AP313 surface display expression vector to form a synthetic bindingsite within CDR3 of a human Fab antibody that would be recognized bygp120. The library of clones produced as described in Example 2 werethen selected or screened for binding to gp120.

For preparing the binding site residues on CD4 specific for gp120, apool of oligonucleotide primers shown below were synthesized with thecomplementary sequence encoding the CD4 amino acid peptide bordered by acoding strand degenerate region ((NNK)₃ where K is G or T and N is A, C,G or T) for encoding 5 amino acids on the amino terminal side of the CD4sequence and 3 amino acids on the carboxy terminal side of the CD4sequence. This region was further bordered by the nucleotide sequencefor encoding the 5′ and 3′ CDR3 amino acid residues in the pC3AP313expression vector. The resulting amplification products had sequencesfor encoding the CD4 sequences specific for binding to gp120 bordered bysequences for randomly encoding 3 amino acids in CDR3.

The PCR reaction resulted in the amplification of the region of theheavy chain fragment in the pC3AP313 phagemid vector clone beginning atframework region 1 (FR1) and extending to the end of the CDR3 which isapproximately 450 base pairs (bp) in length. To amplify the heavy chainregion in PC3AP313, the following primer pairs were used. The 5′oligonucleotide primer, FTX3, having the nucleotide sequence 5′GCAATTAACCCTCACTAAAGGG3′ (SEQ ID NO 54), hybridized to the noncodingstrand of the heavy chain corresponding to the region 5′ of andincluding the beginning of FR1. The 3′ oligonucleotide primer, CD4,having the nucleotide sequence 5′CTCCTCCTCCTCCTCGACGTCMNNMNNMNNCAGAAAACTCCCTTGATTACCMNNMNNMNNMNNMNNACCTCTCGCACAGTAATACACGGC3′ (SEQ ID NO 57) where M is A orC and N is A, C, G or T, hybridized to the coding strand of the heavychain corresponding to CDR3. The oligonucleotide primers weresynthesized by Operon Technologies Alameda, Calif. The expected encodedamino acid residue sequences in CDR3 and flanking framework regionsresulting from the PCR amplification with the 3′ primer have theformula: -NH₂-AVYYCARGXXXXXGNQGSFLXXXDVEEEEE-COOH (SEQ ID NO 58), whereX is Xaa, any amino acid. Refer to the Table of Correspondence and theSequence Listing for the identification of the amino acid residuespresented in single letter format.

The PCR reaction was performed as described in Example 1A. The resultantPCR products were purified, digested with XhoI and AatII and insertedback into the similarly digested pC3AP313 surface display expressionvector for preparation of an expression library as described in Example2.

D. Preparation of Motif Switched CDR3 Synthetic Binding Sites that BindIntegrins

Since the study of integrin function is complicated by native ligandsthat are difficult to work with and often yield data that are difficultto interpret, high affinity, well behaved, integrin ligands have beendesigned in this invention. The first step in this process wasaccomplished by building a synthetic human antibody to target theintegrin ligand binding site as described in the Examples above. Thetarget integrin was a because of its therapeutic importance in tumormetastasis and osteoporosis.

As described in Examples 1A and 1B, Fab antibodies were engineered tobind integrin α_(v)β₃ using a semi-synthetic strategy that involved theinsertion of an RGD motif into the sequence of the heavy chain CDR3 of ahuman antibody. A schematic representation of this process is shown inFIG. 1. In this case, the parent antibody bound to the HIV coat proteingp120. The RGD motif inserted into this CDR was loosely based on the“disintegrin” motif present in numerous snake venoms where the RGD is atthe apex of a disulfide bonded loop. By incorporating a similar motifinto a phage library where the six residues flanking the RGD sequencewere randomized, a series of antibodies were obtained by panning thislibrary on purified α_(v)β₃. Interestingly, these semi-synthetic,RGD-containing antibodies bind α_(v)β₃ nearly 1000-fold better thanα_(v)β₅, and α₅β₁. However, in ligand inhibition studies the selectedantibodies did not distinguish the two β₃-containing integrins, α_(v)β₃and α_(IIb)β₃. Consequently, this invention contemplates the use of themethods as described herein to generate Fab antibodies that exhibitspecificity for each of the two β₃ integrins.

As shown herein, antibody specificity for the ligand binding site ofplatelet integrin α_(IIb)β₃ was obtained through an engineering strategyreferred to as “motif optimization” or “motif switching”. The backbonestructure of one of the previous RGD-containing antibodies, Fab 9, wasused as a scaffold because of its high affinity for the β₃-integrins.The positions corresponding to adhesion motif amino acid residuepositions 5-8 in the heavy chain CDR3 (FIG. 1), that replaces the RGDIamino acid residue sequence in HCDR3 of Fab 9 (SEQ ID NO 4 from positionnumber 7 to 10), were randomized. Optimal binding motifs specific forα_(IIb)β₃ were selected by panning the new phage library on purifiedα_(IIb)β₃, also referred to as gpIIb/IIIa, as described in Example 3. Asshown below, the selected antibodies do not contain the RGD sequence,but all still block function, i.e., the binding of ligands togpIIb/IIIa. These synthetic anti-integrin antibodies have great promisefor simplifying the study of the integrin ligand binding pocket and alsofor inhibiting platelet activity in vivo. Since peptide ligands for manyother integrins have been identified, the methods of this invention areuseful for synthesizing highly specific antibodies that bind the ligandbinding pocket of other members of this protein family.

In order to obtain antibodies that exhibited a higher affinity to theselected receptors and have greater sequence heterogeneity, a secondcycle of mutagenesis was performed on a RGD-containing monoclonalantibody obtained as described in Example 1B. For this mutagenesisprocedure, referred to as CDR motif switching or motif optimization, Fab9 (SEQ ID NO 4) that exhibited the highest affinity towards α_(v)/β₃ andgpIIb/IIIa was selected as the motif scaffold for incorporating newbinding sites.

An oligonucleotide was designed for use in PCR amplification of the Fab9 heavy chain that would result in the mutagenesis of the amino acidresidue sequence in Fab 9, RGDI, beginning at position 7 and ending atposition 10 in SEQ ID NO 4. This region corresponded to amino acidresidue 5-8 in the heavy chain CDR3 as shown in FIG. 1. Thus, theoligonucleotide was randomized for 12 nucleotides with the triplet codonsequence MNN. The complementary nucleotide would contain the tripletcodon NNK written in the 5′ to 3′ direction. The oligonucleotide asshown below was also designed to retain the nucleotide sequencebordering the RGDI sequence such that the resulting motif switchedantibodies would only contain mutations in the RGDI site and not in theflanking regions. In addition, the oligonucleotide contained sequencesthat were from the framework 3 and 4 regions bordering the heavy chainCDR3 to maintain that portion of the nucleotide sequence of Fab 9.

Thus, for preparing motif optimized binding sites within CDR3 of a humanFab antibody of this invention, specifically Fab 9, a pool ofoligonucleotide primers shown below were synthesized with a codingstrand degenerate region ((NNK)₃ where K is G or T and N is A, C, G orT) for encoding 4 mutated amino acids flanked by Fab 9 CDR3 amino acidsequences as listed in SEQ ID NO 4. This region was further bordered bythe nucleotide sequence for encoding the flanking amino acid residues inthe pMT12 expression vector that was present in Fab 9.

The PCR reaction resulted in the amplification of the region of theheavy chain fragment in the pMT12 phagemid vector clone beginning atframework region 1 (FR1) and extending to the end of the CDR3 domainwhich is approximately 450 base pairs (bp) in length. To amplify thisregion, the following primer pairs were used. The 5′ oligonucleotideprimer, FTX3, having the nucleotide sequence 5′ GCAATTAACCCTCACTAAAGGG3′(SEQ ID NO 54), hybridized to the noncoding strand of the heavy chaincorresponding to the region 5′ of and including the beginning of FR1.The 3′ oligonucleotide primer, MOTIF, having the nucleotide sequence 5′CTCCTCCTCCTCCTCGACGTCCATATAATAGCAATTCTMNNMNNMNNMNNCCCAAACGAGCACCCCACTCTCGCACAATAATA3′ (SEQ ID NO 52) where M is A or C andN is A, C, G or T, hybridized to the coding strand of the heavy chaincorresponding to the CDR3 Fab 9. The oligonucleotide primers weresynthesized by Operon Technologies, Alameda, Calif. The expected encodedamino acid residue sequences in CDR3 resulting from the PCRamplification with the 3′ primer have the formula:-NH₂-VGCSFGXXXXRNCYYMDV-COOH (SEQ ID NO 67), where X is Xaa, any aminoacid. Refer to the Table of Correspondence and the Sequence Listing forthe identification of the amino acid residues presented in single letterformat.

The PCR reaction was performed as described in Example 1A. The resultantPCR products were purified, digested with XhoI and AatII and insertedinto the similarly digested pC3AP313 surface display expression vectorfor preparation of an expression library as described in Example 2. Theamino acid residue sequences resulting from the selection of motifswitch CDR3 antibodies of this invention are described below in Examples4 and 5 and are listed in the Section entitled “Binding SitePolypeptides”.

2. Production of Phagemid Fab-displayed Synthetic Binding Sites

In practicing this invention to obtain expression of Fab-displayproteins containing a synthetic binding site on a phage surface, theheavy (Fd consisting of V_(H) and C_(H)1) and light (kappa) chains(V_(L), C_(L)) of antibodies were first targeted to the periplasm of E.coli for the assembly of heterodimeric Fab molecules.

In this system, the first cistron encoded a periplasmic secretion signal(pelB leader) operatively linked to the fusion protein, Fd-cpIII. Thesecond cistron encoded a second pelB leader operatively linked to akappa light chain. The presence of the pelB leader facilitated thecoordinated but separate secretion of both the fusion protein containingthe synthetic binding site and light chain from the bacterial cytoplasminto the periplasmic space.

In this process, each chain was delivered to the periplasmic space bythe pelB leader sequence, which was subsequently cleaved. The heavychain containing the synthetic binding was anchored in the membrane bythe cpIII membrane anchor domain while the light chain was secreted intothe periplasm. Fab molecules were formed from the binding of the heavychain with the soluble light chains.

A. Preparation of a Dicistronic Expression Vector, pComb3, Capable ofExpressing a Phagemid Fab Display Protein

A schematic of the pComb3 phagemid expression vector for use inexpressing the synthetic binding site-containing antibodies of thisinvention is shown in FIG. 2. The antibody Fd chain comprising variable(V_(H)) and constant (C_(H)1) domains of the heavy chain were fused withthe C-terminal domain of bacteriophage gene III (3) coat protein. GeneIII of filamentous phage encodes a 406-residue minor phage coat protein,cpIII (cp3), which is expressed prior to extrusion in the phage assemblyprocess on a bacterial membrane and accumulates on the inner membranefacing into the periplasm of E. coli.

The phagemid vector, designated pComb3, allowed for both surface displayand soluble forms of Fabs. The vector was designed for the cloning ofcombinatorial Fab libraries. The XhoI and SpeI site were provided forcloning complete PCR-amplified heavy chain (Fd) sequences consisting ofthe region beginning with framework 1 and extending through framework 4.An AatII restriction site is also present but not identified in FIG. 2.The presence of the AatII site allows for the insertion of XhoI/AatIIdigests of the PCR products prepared in Example 1 that contain sequencesbeginning with framework 1 and extending to the end of the CDR3 domainin which the sequences for encoding the synthetic binding sites arelocated. The insertion of an XhoI/AatII digest into pC3AP313 results inthe fusion of the insert with the framework 4 domain in the pC3AP313vector. Thus, the insertion results in the in-frame ligation of acomplete heavy chain fragment consisting of PCR amplified framework 1through CDR3 and retained pC3AP313 retained framework 4. The SacI andXbaI sites were provided for cloning PCR amplified antibody lightchains. The cloning sites were compatible with previously reported mouseand human PCR primers as described by Huse et al., Science,246:1275-1281 (1989) and Persson et al., Proc. Natl. Acad. Sci., USA,88:2432-2436 (1991). The nucleotide sequence of the pelB, a leadersequence for directing the expressed protein to the periplasmic space,was as reported by Huse et al., supra.

The vector also contained a ribosome binding site as described by Shineet al., Nature, 254:34 (1975). The sequence of the phagemid vector,pBluescript, which includes ColE1 and F1 origins and a beta-lactamasegene, has been previously described by Short et al., Nuc. Acids Res.,16:7583-7600 (1988) and has the GenBank Accession Number 52330 for thecomplete sequence. Additional restriction sites, SalI, AccI, HincII,ClaI, HindIII, EcoRV, PstI and SmaI, located between the XhoI and SpeIsites of the empty vector were derived from a 51 base pair stufferfragment of pBluescript as described by Short et al., supra. Anucleotide sequence that encodes a flexible 5 amino acid residue tethersequence which lacks an ordered secondary structure was juxtaposedbetween the Fab and cp3 nucleotide domains so that interaction in theexpressed fusion protein was minimized.

Thus, the resultant combinatorial vector, pComb3, consisted of a DNAmolecule having two cassettes to express one fusion protein, Fd/cp3, andone soluble protein, the light chain. The vector also containednucleotide residue sequences for the following operatively linkedelements listed in a 5′ to 3′ direction: a first cassette consisting ofLacZ promoter/operator sequences; a NotI restriction site; a ribosomebinding site; a pelB leader; a spacer region; a cloning region borderedby 5′ XhoI and 3′ SpeI restriction sites; the tether sequence; thesequences encoding bacteriophage cp3 followed by a stop codon; a NheIrestriction site located between the two cassettes; a second lacZpromoter/operator sequence followed by an expression control ribosomebinding site; a pelB leader; a spacer region; a cloning region borderedby 5′ SacI and a 3′ XbaI restriction sites followed by expressioncontrol stop sequences and a second NotI restriction site.

In the above expression vector, the Fd/cp3 fusion and light chainproteins were placed under the control of separate lac promoter/operatorsequences and directed to the periplasmic space by pelB leader sequencesfor functional assembly on the membrane. Inclusion of the phage F1intergenic region in the vector allowed for the packaging ofsingle-stranded phagemid with the aid of helper phage. The use of helperphage superinfection allowed for the expression of two forms of cp3.Consequently, normal phage morphogenesis was perturbed by competitionbetween the Fd/cp3 fusion and the native cp3 of the helper phage forincorporation into the virion. The resulting packaged phagemid carriednative cp3, which is necessary for infection, and the encoded Fab fusionprotein, which is displayed for selection. Fusion with the C-terminaldomain was necessitated by the phagemid approach because fusion with theinfective N-terminal domain would render the host cell resistant toinfection.

The pComb3 expression vector described above forms the basic constructof pC3AP313, p7EIII, and pMT12 Fab display phagemid expression vectorsused in this invention for the production of human Fab antibodiescontaining synthetic binding sites. The pC3AP313 and p7EIII phagemidexpression vectors described in Example 1 having the respective ATCCaccession numbers, 75408 and 75409, are pComb3-based vectors containingheavy and light chain sequences for encoding human Fab antibodiesagainst tetanus toxin. The pMT12 phagemid expression vector, while basedon pComb3, has been digested with NheI and SpeI to delete the sequencethat encodes the bacteriophage coat protein 3, thereby resulting in theexpression of soluble human anti-gp120 Fab. The pMT12 phagemid wasdeposited with ATCC in a bacterial host and has the accession number69079.

B. Preparation of Expression Vector Libraries for the Expression of thePhagemid Fab-display Proteins

1) Phagemid Library Construction

In order to obtain expressed human Fab antibodies having both heavy andlight chain fragments, phagemid libraries were constructed. Thelibraries provided for the expression of recombinant human Fabantibodies having heavy and light chains where the synthetic bindingsites of this invention are displayed in the heavy chain CDR3. The PCRproducts resulting from each of the amplification reactions prepared inExample 1 were separately inserted into a phagemid expression vector toprepare phagemid libraries. As described below, the resultant gelpurified heavy chain PCR fragments prepared in Examples 1A-1D weredigested with the restriction enzymes and separately ligated into thepC3AP313 phagemid expression vector that was similarly digested.

For preparation of phagemid libraries for expressing the PCR productsprepared in Examples 1A-1D, the PCR products were separately digestedwith XhoI and AatII and separately ligated with a similarly digestedpC3AP313 phagemid expression vector prepared as described in Examples 1and 2A. The ligation resulted in operatively linking the framework 1through CDR3 PCR products with the framework 4 domain present in thepC3AP313 vector.

Phagemid libraries for expressing each of the Fab display syntheticbinding sites of this invention were prepared in the followingprocedure. To form circularized vectors containing the PCR productinsert, 640 ng of the digested PCR products were admixed with 2 ug ofthe linearized pC3AP313 phagemid vector and ligation was allowed toproceed overnight at room temperature using 10 units of BRL ligase(Gaithersburg, Md.) in BRL ligase buffer in a reaction volume of 150 ul.Five separate ligation reactions were performed to increase the size ofthe phage library having synthetic binding site CDR3 regions. Followingthe ligation reactions, the circularized DNA was precipitated at −20 Cfor two hours by the admixture of 2 ul of 20 mg/ml glycogen, 15 ul of 3M sodium acetate at pH 5.2 and 300 ul of ethanol. DNA was then pelletedby microcentrifugation at 4 C for 15 minutes. The DNA pellet was washedwith cold 70% ethanol and dried under vacuum. The pellet was resuspendedin 10 ul of water and transformed by electroporation into 300 ul of E.coli XL1-Blue cells to form a phage library. The total yield from thePCR amplification and transformation procedure described herein wasapproximately 5×10⁷ transformants for each library with the exceptionthat the motif optimized library, prepared from the PCR productsdescribed in Example 1D, contained 3×10⁷.

After transformation, to isolate phage on which Fabs displayingsynthetic binding sites been induced for subsequent panning on targetantigens such as the integrin receptors α_(v)β₃, α_(v)β₅, αIIb/β₃, andgp120 protein, 3 ml of SOC medium (SOC was prepared by admixture of 20grams (g) bacto-tryptone, 5 g yeast extract and 0.5 g NaCl in 1 liter ofwater, adjusting, the pH to 7.5 and admixing 20 ml of glucose justbefore use to induce the expression of the Fd-cpIII and light chainheterodimer) were admixed and the culture was shaken at 220 rpm for 1hour at 37 C, after which 10 ml of SB (SB was prepared by admixing 30 gtryptone, 20 g yeast extract, and 10 g Mops buffer per liter with pHadjusted to 7) containing 20 ug/ml carbenicillin and 10 ug/mltetracycline and the admixture was shaken at 300 rpm for an additionalhour. This resultant admixture was admixed to 100 ml SB containing 50ug/ml carbenicillin and 10 ug/ml tetracycline and shaken for 1 hour,after which helper phage VCSM13 (10¹² pfu) were admixed and theadmixture was shaken for an additional 2 hours.

After this time, 70 ug/ml kanamycin was admixed and maintained at 30 Covernight. The lower temperature resulted in better heterodimerincorporation on the surface of the phage. The supernatant was clearedby centrifugation (4000 rpm for 15 minutes in a JA10 rotor at 4 C).Phage were precipitated by admixture of 4% (w/v) polyethylene glycol8000 and 3% (w/v) NaCl and maintained on ice for 30 minutes, followed bycentrifugation (9000 rpm for 20 minutes in a JA10 rotor at 4 C). Phagepellets were resuspended in 2 ml of PBS and microcentrifuged for threeminutes to pellet debris, transferred to fresh tubes and stored at −20 Cfor subsequent screening as described below.

For determining the titering colony forming units (cfu), phage (packagedphagemid) were diluted in SB and 1 ul was used to infect 50 ul of fresh(A_(O0600)=1) E. coli XL1-Blue cells grown in SB containing 10 ug/mltetracycline. Phage and cells were maintained at room temperature for 15minutes and then directly plated on LB/carbenicillin plates.

3. Selection of the Phagemid Fab-Displayed Synthetic Binding SiteProteins

A. Multiple Pannings of the Phage Library Having Phagemid Fab-displayedSynthetic Binding Site Proteins

The phage libraries produced in Example 2 having heavy chain fragmentswith Fab display synthetic binding site regions were panned as describedherein on microtiter plates coated with selected target molecules. Thetarget molecules used in screening the phagemid-anchored Fab-displayedsynthetic binding site proteins included the following: purifiedvitronectin receptor (also referred to as VNR) comprised by the pairs ofintegrin subunits alpha v/beta 3 (α_(v)β₃), alpha v/beta 5 (α_(v)β₅) orαIIb/β₃ (gpIIb/IIIa); and the HIV glycoprotein, gp120.

All the vitronectin receptors are commercially for available from TeliosPharmaceutical, San Diego, CA. Alternatively, the α_(v)/β₃ and α₃/β₅VNRs were purified as described by Smith et al., J. Biol. Chem.,265:11008-11013 (1990), the disclosure of which is hereby incorporatedby reference. Briefly, human placentas were obtained, cut into smallpieces and extracted by incubation with 100 mM octyl glucose, 2 mMCaCl₂, 1 mM phenylmethylsulfonyl fluoride in PBS for 30-40 minutes atroom temperature. After filtering the extract through gauze andcentrifugation at 12,000×g for 27 minutes, the supernatant was pumpedover an anti-VNR antibody Affi-gel column (Bio-Rad, Richmond, Calif.) inwhich an antibody specific for the particular VNR subunit was previouslycoupled according to manufacturer's instructions. A monoclonal antibody,LM 609, described by Cheresh et al., J. Biol. Chem., 262:17703-17711(1987), was used in the purification of α_(v)/β₃. An antibody, LM 142,described by Cheresh et al., Cell, 57:59-69 (1989), was used in thepurification of α_(v)/β₅. In addition, antibodies against all 3 VNRs arecommercially available for use in purifying target molecules used inscreening for Fab-displayed synthetic binding sites of this invention.

After the placenta extract was pumped through the anti-α_(v)/β₃ column,the flow through was collected for application on an anti-α_(v)/β₅column. The column was then washed with 50 column volumes of PBS, 0.1%NONIDET P-40, 2 mM CaCl₂ and 50 column volumes of 0.01 M acetic acid atpH 4.5 containing 0.1% NP-40 and 2 mM CaCl₂. VNR having the α_(v)/β₅subunits was eluted with 0.01 M acetic acid at pH 3.0 containing 0.1%NP-40 and 2 mM CaCl₂. The flow through was then applied to theanti-α_(v)/β₅ SEPHAROSE column (Pharmacia, Piscataway, N.J.) preparedaccording to manufacturer's instructions to isolate the specific VNRreceptor. The VNR was eluted with 0.01 M acetic acid at pH 3.0containing 1 mM of CaCl₂, MnCl₂ and 0.1% NP-40. The fractions from bothcolumns were neutralized by addition of 1.0 ml of 3.0 M Tris-HCl at pH8.8. The VNR having the αIIb/α₃ subunits (also referred to asgpIIb/IIIa) was isolated according to the procedure in U.S. Pat. No.5,114,842. The above-purified VNRs were used in the panning proceduredescribed below at a concentration of 1 ug/well in a coating bufferconsisting of 20 mM Tris-HCl at pH 7.4, 150 mM NaCl and 1 mM each ofCaCl₂, MgCl₂ and MnCl₂.

The recombinant gp120 of HIV-1 strain IIIb was commercially availablefrom American Biotechnologies, Ossining, N.Y. The reagent was used at aconcentration of 40 ug/ml in the coating buffer, 0.1 M bicarbonate at pH8.6.

The panning procedure described was a modification of that originallydescribed by Parmley et al., Gene, 73:305-318 (1988). Two to four wellsof a microtiter plate (Costar 3690) were coated overnight at 4 C withthe purified target proteins (VNRs and gp120) prepared above. The wellswere washed twice with water and blocked by completely filling the wellwith 3% (w/v) bovine serum albumin (BSA) in PBS and incubating the plateat 37 C for 1 hour. Blocking solution was removed by shaking, 50 ul ofthe phage library prepared above (typically 1011 cfu) were added to eachwell, and the plate was incubated for 2 hours at 37 C.

Phage were removed and the plate was washed once with water. Each wellwas then washed 10 times with TBS/TWEEN (50 mM Tris-HCl at pH 7.5, 150mM NaCl, 0.5% TWEEN 20) over a period of 1 hour at room temperature thenpipetted up and down to wash the well, each time allowing the well toremain completely filled with TBS/TWEEN between washings. The plate waswashed once more with distilled water and adherent phage were eluted bythe addition of 50 ul of elution buffer (0.1 M HCl, adjusted to pH 2.2with solid glycine, containing 1 mg/ml BSA) to each well and incubationat room temperature for 10 minutes. The elution buffer was pipetted upand down several times, removed, and neutralized with 3 ul of 2 M Trisbase per 50 ul of elution buffer used. Eluted phage were used to infect2 ml of fresh (OD₆₀₀=1) E. coli XL1-Blue cells for 15 minutes at roomtemperature, after which 10 ml of SB containing 20 ug/ml carbenicillinand 10 ug/ml tetracycline was admixed. Aliquots of (20, 10, and 1/10 ulwere removed for plating to determine the number of phage (packagedphagemids) that were eluted from the plate. The culture was shaken for 1hour at 37 C, after which it was added to 100 ml of SB containing 50ug/ml carbenicillin and 10 ug/ml tetracycline and shaken for 1 hour.Helper phage VCSM13 (10¹² pfu) were then added and the culture wasshaken for an additional 2 hours. After this time, 70 ug/ml kanamycinwas added and the culture was incubated at 37 C overnight. Phagepreparation and further panning were repeated as described above.

To select antibodies with specificity for each integrin from the phagelibrary, integrins α_(v)β₃ or α_(IIb)β₃ were immobilized in Costar 3690microtiter plates in 50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂and 1 mM MnCl₂. Selection of phage bearing antigen-specific antibodyfragments was performed by panning for six rounds as described above,but with the following modification. In order to select for antibodieswith specificity for one or the other integrin, the second integrin wasused as a soluble competitor at 20 ug/ml during panning of the phagewith the immobilized target integrin. For example, to select antibodiesspecific for α_(IIb)β₃, this receptor was immobilized in microtiterwells and α_(IIb)β₃ was added, in solution, to the phage library duringthe panning step.

Following each round of panning, the percentage yield of phage weredetermined, where % yield−(number of phage eluted/number of phageapplied)×100.

As an alternative to elution with acid, phage bound to the wells of themicrotiter plate were eluted by admixing 50 ul of a solution of 10⁻⁵M ofthe particular purified coat protein prepared above diluted in PBSfollowed by a maintenance period of 1 hour at 37 C. The solution wasthen pipetted up and down to wash the wells. The resultant eluate wastransferred to 2 ml of fresh E. coli XL1-Blue cells for infection asdescribed above for preparing phage and further panning.

The final phage output ratio was determined by infecting 2 ml oflogarithmic phase XL1-Blue cells as described above and plating aliquotson selective plates. In the first panning, approximately 10¹¹ phage wereapplied to four wells and approximate yields ranged from 5.0 to 8.0×10⁵eluted phage. After the fourth panning, eluted phage ranged from 1.0×10⁶to 1.0×10⁸ phage. The motif switched CDR3 antibodies with their aminoacid residue sequences listed in SEQ ID NOs from 19-24 were obtainedafter five rounds of panning on gpIIb/IIIa. The remainder of thegpIIb/IIIa-reactive clones listed in SEQ ID NOs 25-34, after 10 roundsof panning to select for higher affinity binders that had greater aminoacid sequence heterogeneity. From this procedure, clones were selectedfrom each of the Fab libraries for their ability to bind to theirrespective selected target proteins. The panned phage surface librarieswere then converted into ones expressing soluble Fab-displayed syntheticbinding site proteins for further characterization as described inExamples 4 and 5.

B. Preparation of Soluble Fab-displayed Binding Site Proteins

In order to further characterize the specificity of the Fab-displayedsynthetic binding site proteins expressed on the surface of phage asdescribed above, soluble heterodimers were prepared and analyzed inELISA assays on target-coated plates and by competitive ELISA withincreasing concentrations of soluble competitor protein as describedbelow.

To prepare soluble Fabs consisting of heavy and light chains (i.e.,heterodimers), phagemid DNA from positive clones selected in Example 2Aabove was isolated and digested with SpeI and NheI. Digestion with theseenzymes produced compatible cohesive ends. The 4.7 kb DNA fragmentlacking the gIII portion was gel-purified (0.6% agarose) andself-ligated. Transformation of E. coli XL1-Blue afforded the isolationof recombinants lacking the gIII fragment. Clones were examined forremoval of the gIII fragment by XhoI/XbaI digestion, which yielded an1.6 kb fragment.

Clones were then grown in 100 ml SB containing 50 ug/ml carbenicillinand 20 mM MgCl₂ at 37 C until an OD₆₀₀ of 0.2 was achieved. IPTG (1 mM)was added and the culture grown overnight at 30 C (growth at 37 Cprovides only a light reduction in heterodimer yield). Cells werepelleted by centrifugation at 4000 rpm for 15 minutes in a JA10 rotor at4 C. Cells were resuspended in 4 ml PBS containing 34 ug/mlphenylmethylsulfonyl fluoride (PMSF) and lysed by sonication on ice (2-4minutes at 50% duty). Debris was pelleted by centrifugation at 14,000rpm in a JA20 rotor at 4 C for 15 minutes. The supernatant was useddirectly for ELISA analysis and was stored at −20 C. For the study of alarge number of clones, 10-ml cultures provided a sufficient amount ofFab-displayed synthetic binding site proteins for analysis. In thiscase, sonications were performed in 2 ml of buffer.

The soluble heterodimers prepared above were assayed by ELISA whereapplicable as described in Example 4 and 5.

4. Characterization of Soluble Fab-RGD and Gp120 Specific Binding SiteProteins

A. Determination of Specificity of the Binding Site Proteins

1) ELISA

Three major criteria for a probe of an integrin ligand binding site arethe following: 1) The antibody must block the binding of the naturalligand for the receptor; 2) RGD-peptides should block the binding of theantibody; and 3) The binding between the antibody and the integrinshould be divalent cation-dependent. Preliminary ELISA assays wereperformed to first characterize the binding specificity of the pannedphage monoclonal antibodies prepared above toward related integrins. ForELISA, 1 ug/well of the purified VNRs prepared in Example 3A wasseparately admixed to individual wells of a microtiter plate andmaintained at 4 C overnight to allow the protein solution to adhere tothe walls of the well. After the maintenance period, the wells werewashed once with PBS and thereafter maintained with a solution of 3% BSAto block nonspecific sites on the wells. The plates were maintained at37 C for 1 hour after which time the plates were inverted and shaked toremove the BSA solution. Soluble Fab heterodimers expressing thesynthetic binding site proteins-prepared in Example 3B were then admixedseparately to each well and maintained at 37 C for 1 hour to form aimmunoreaction products. Following the maintenance period, the wellswere washed 10 times with PBS to remove unbound soluble antibody andthen maintained with a secondary goat anti-human FAB conjugated toalkaline phosphatase diluted in PBS containing 1% BSA. The wells weremaintained at 37 C for 1 hour after which the wells were washed 10 timeswith PBS followed by development with p-nitrophenyl phosphate.

The results of the ELISA assays showed that the Fab-displayed RGDC2binding site (Mab 8, also referred to as VnRβ₃) as listed in SEQ ID NO 8bound specifically to α_(v)/β₃ and not to α_(v)/β₅ as compared tobackground binding with BSA. Similarly, the Fab-displayed RGDC2 bindingsite (Mab 11, also referred to as VnRβ₅) as listed in SEQ ID NO 11 boundspecifically to α_(v)/β₅ and not to α_(v)/β₃ as compared to backgroundbinding with BSA. Thus, the these two selected Fab-displaying RGDbinding site proteins of this invention exhibited specificity to one VNRreceptor and not another depending of the subunit composition. Thisspecificity has not been demonstrated before this invention with aRGD-containing sequence for a particular receptor.

In addition, with ELISA performed on gpIIB/IIIa-coated surfaces preparedas described for VnR-coated surfaces, the selected Fab-displaying RGDC2binding site protein (MAb 19, also referred to as IIb/IIIa-19) as listedin SEQ ID NO 3 was shown to be specific for gpIIb/IIIa and notcross-react with the other VNR receptors. Thus, the method of preparingFab-displayed synthetic binding site proteins allows for the productionof binding site molecules that exhibit heightened specificity for singletarget molecules as shown herein.

Another monoclonal antibody designated Fab 9 (also referred to as Mab 9,as VnRB3-5 and has the CDR3 amino acid residue sequence in SEQ ID NO 4)was prepared in soluble form and evaluated for its ability to inhibitthe binding of vitronectin, fibronectin and fibrinogen to both α_(v)/β₃and gpIIb/IIIa receptors. Fab 9 blocked the binding of the highestaffinity ligand for its respective receptor, namely vitronectin forα_(v)/β₃ and fibrinogen for gpIIb/IIIa. In the same assay, Fab 9 failedto block vitronectin binding to α_(v)/β₅ with any appreciable affinity.Thus, Fab 9 exhibited unique specificity in that α_(v)/β₅ has an α_(v)subunit and the β₅ subunit is 55% identical to β₃. Therefore, theappropriate display of the RGD sequence with CDR effectively programmedreceptor specificity into the antibody. All Fabs produced by the methodsof this invention, including Fab 9, competed with vitronectin for thebinding to the receptor in subnanomolar range with IC₅₀'s ranging from1×10⁻¹⁰ to 5×10⁻¹⁰. The most potent snake venoms in contrast competeonly in the nanomolar range. The affinity of Fab 9 was furthercharacterized by surface plasmon resonance, the procedure of which isdescribed in Example 5 to yield a K_(d) of 2.5×10⁻¹⁰ M.

2) Cell Adhesion Assays and Competitive ELISA

Immunoreactive Fabs as determined in the above ELISAs were then analyzedby cell adhesion assays and by competition ELISA to determine theaffinity of the Fab-displayed synthetic binding site proteins. In celladhesion assays, receptor clustering can increase cellular avidity andcells have the potential to metabolize inhibitors. These conditions canoverride the effects of a weak inhibitor. The cell adhesion assay wasperformed as described by Wayner et al., J. Cell. Biol., 113:919-929(1991) and Felding-Habermann et al., J. Biol. Chem., 267:5070-5077(1992), the disclosures of which are hereby incorporated by reference.

a) Assays with RGD-containing Fabs

The assay was performed as described above for ELISA with the exceptionthat increasing concentrations of soluble anti-VNR antibodies were addedto compete the binding of the RGD-specific VNR receptors on the cellsurface to the RGD-containing Fab (also referred to as Fab-displayedRGD) coated on the plate.

For experiments where the binding of cells to the Fab-displayed RGD orcyclized RGD (RGDC2) was inhibited by antibodies to α_(v)/β₃, the cellsused in the assays were M21 human melanoma cells as they expressα_(v)/β₃ VNR. For experiments where the binding of the cells to theFab-displaying RGD or cyclized RGD (RGDC2) was inhibited by antibodiesto α_(v)/β₅, the cells used in the assays were UCLA-P3 lung carcinomacells as they express α_(v)/β₅ VNR. Other cells expressing the VNRs arecontemplated for use in the characterization of selected Fab-displayedsynthetic binding sites depending on the target molecule. The competingantibodies ranged in concentration from 10⁻⁹ M up to 10⁻⁶ M inconcentration admixed in the presence of the soluble RGD binding sitecontaining Fabs.

In the above inhibition of cell adhesion assay, maximal inhibition ofbinding was achieved at a concentration of 10⁻⁶ M of competinganti-α_(v)/β₃ while an anti-HIV was ineffective.

When Fab 9 that contained an RGD binding site was used in cell adhesionassays, it blocked the melanoma cell adhesion to immobilized vitronectinwith an IC₅₀ of 4 nM in contrast to the framework antibody pMT12 thathad no effect on cell adhesion. More importantly, Fab 9 did notinterfere with adhesion to fibronectin, an event largely mediatedthrough β1 integrins. This finding provides further evidence that Fab 9is specific to beta 3 integrins. In similar assays, Fab 9 was comparedto vitronectin in the ability to support cellular adhesion. Bothproteins supported the binding of cells saturating at approximately 5ug/ml of Fab or vitronectin. Vitronectin was only slightly moreeffective at promoting the adhesion of melanoma cells.

Additional competition ELISA assays were performed to confirm thespecificity of the Fab-displayed RGD binding site. In these assays, thepurified receptors α_(v)/β₅ and α_(v)/β₃ were separately coated on theplate at a concentration of 1 mM. One nM of the soluble Fab-displayedRGD-binding site shown to be specific for α_(v)/β₃ by ELISA as shownabove was admixed with iodinated soluble vitronectin (TeliosPharmaceuticals) and then added to the receptor-coated wells. Theα_(v)/β₃-specific Fab-displayed RGD binding site completely inhibitedthe binding of labelled vitronectin to the wells while the Fab-displayedRGD binding site specific for α_(v)/β₅ did not completely inhibit thebinding of labelled vitronectin even at 10⁻⁶ M concentration. Thus, theFab-displayed RGD binding site proteins of this invention exhibitheightened specificity towards specific VNRs depending on their subunit.

In a separate competition assay, the binding of radiolabeled Fabs, forexample Fab 9, to α_(b)/β₃ and gpIIb/IIIa was competed by RGD-containingpeptides and not by the RGE-containing inactive analog. Moreover, thebinding of Fab 9 to α_(v)/β₃ and gpIIb/IIIa was shown to be divalentcation-dependent as assessed by the binding of radiolabeled Fab 9 toα_(v)/β₃ in the purified receptor binding assay performed as describedabove. Immobilized α_(v)/β₃ was first depleted of endogenously bounddivalent cations by treatment with EDTA. The receptor was thenreplenished with 0.5 mM of either calcium, manganese, or magnesium. Eachof these ions was able to support Fab 9 binding to the receptor. Nearlyidentical results were obtained in similar assays performed withgpIIb/IIIa receptor. Collectively, the results obtained from assaysperformed with Fab 9, having the RGD binding site as listed in SEQ ID NO4, show that Fab 9 binds the ligand binding pocket of α_(v)/β₃ andgpIIb/IIIa with high affinity and specificity.

Other assays were performed to demonstrate specificity of theFab-displayed RGD binding site proteins of this invention. A gpIIb/IIIasandwich ELISA assay was performed on 10 ug/ml (100 ul) offibrinogen-coated plates. After blocking the plate as described above,the RGD-expressing, Fab, Mab 19, was admixed with purified gpIIb/IIIareceptor. After allowing the RGD-expressing Fab and receptor admixtureto immunoreact with the fibrinogen, the wells were washed and the amountof bound gpIIb/IIIa was determined using an anti-IIIa antibody. Theresults of this assay showed that the Mab 19 expressing an RGDC2 bindingsite blocked the binding of gpIIb/IIIa to fibrinogen in a dose-dependentmanner where maximal inhibition was seen with 5 uM of Fab. No effect wasseen with a control anti-gp120 Fab.

b) Assays with Fabs Having the Gp120 Binding Site

Competitive ELISA using soluble CD4 to compete the binding of the Fabsdisplaying the CD4 sequence recognized by gp120 are then analyzed bycompetition ELISA to determine the affinity of the selectedheterodimers. The ELISA is performed as described above on microtiterwells separately coated with 5 ug/ml of gp120 (American Biotechnologies)in 0.1 M bicarbonate buffer at pH 8.6. Increasing concentrations ofsoluble or free CD4 (American Biotechnologies) ranging in concentrationfrom 10⁻¹¹ M up to 10⁻⁷ M diluted in 0.5% BSA/0.025% TWEEN 20/PBS areadmixed with the soluble Fabs displaying the CD4 sequence, the dilutionsof which are determined in titration experiments that resulted insubstantial reduction of OD values after a 2-fold dilution. The platesare maintained for 90-120 minutes at 37 C and carefully washed ten timeswith 0.05% TWEEN 20/PBS before admixture of alkalinephosphatase-labelled goat anti-human IgG F(ab′)2 at a dilution of 1:500followed by maintenance for 1 hour at 37 C. Development was performed asdescribed for ELISA.

3) Competition Assays with Peptides Derived from RGD-containing FabAntibodies

Additional competition assays were performed with a peptide sequencederived from Fab 9. With the methods of this invention, engineeringsemi-synthetic antibodies by manipulating HCDR3 offered a unique vehiclefor the expression and analysis of random peptide sequences. Thesesequences can be incorporated into the antigen binding site, whereconformational constraints may provide peptide motifs with additionalstructure. Small peptides derived from CDR sequences of antibodiesraised via immunization have been shown to emulate whole antibodies bothin terms of binding activity and in the ability to block proteinfunction as described by Levi et al., Proc, Natl. Acad. Sci., USA,90:4374-4378 (1993) and Welling et al., J. Chromatoar., 548:235-242(1988). However, the binding affinity of such peptides is generally 2-3orders of magnitude below the parent antibody from which they arederived.

To determine if the CDR was an appropriate vehicle for displaying andidentifying inhibitory peptide motifs, the ability of synthetic peptidesderived from the sequence of Fab 9 to block ligand binding to α_(v)β₃,α_(v)β₅ and α_(IIb)β₃ was measured. One peptide synthesized as describedbelow for use in this assay contained a RGD motif positioned on the apexof the CDR3 loop but lacked the remaining flanking regions of the CDR3and rest of the heavy chain. The sequence of the peptide is listed inSEQ ID NO 12. A peptide having the same sequence but flanked by glycineresidues to prevent cyclization was also prepared and has the amino acidresidue sequence in SEQ ID NO 68. A longer peptide, having the aminoacid residue sequence in SEQ ID NO 4 containing all of the amino acidsin SEQ ID NO 12 along with flanking amino acid residues, was alsoprepared. These assays were performed in order to assess the ability ofthis particular peptide to inhibit the binding of a high affinity ligandto a receptor, namely the binding of vitronectin to α_(v)/β₃ or α_(v)/β₅and fibrinogen to gpIIb/IIIa.

The RGD motif originally inserted into the CDR contained two flankingcysteine residues to provide the opportunity for constraining theflexibility of the RGD loop by the formation of a disulfide bond. Sinceit was not evident whether this disulfide bond actually formed in theantibody's CDR, the potential conformations of the adhesion motif wereemulated in synthetic peptides by generating both cyclic and linearforms of each peptide cyclization was achieved and confirmed asdescribed herein.

Peptides derived from HCDR3 sequences were synthesized on Rink's amideresin (0.5 mmol/gm, NovaBiochem) with an Advanced Chemtech 350 multiplepeptide synthesizer using Fmoc synthesis as described by Fields et al.,Int. J. Peptide Protein Res., 35:161-214 (1990). Amino acids werecoupled with diisopropylcarbodiimide and 1-hydroxybenzotriazolemonohydrate. The side chain Fmoc amino acids were protected by Pmc forarginine, trityl for Cys and butyl for Ser, Thr Asp and Tyr. Eachpeptide was blocked at the N-terminus by acetylation and at theC-terminus by amidation. Peptides were cleaved from the resin with 10%trifluoroacetic acid/dichloromethane and protecting groups were removedwith 5% water/5% anisole/2.5% ethanedithiol/7.5% phenol intrifluoroactetic acid (45). Peptides were precipitated with diethylether and purified on a C-18 column (Vydac 201TP1022, 2.2×25 cm) with a0-100% water/acetonitrile gradient containing 1% trifluoroacetic acid.Cyclizations of cysteine peptides (1 mg/ml) were carried to completionin 20% DMSO/H₂O at pH 4.0 in 1 day as described by Tam et al., J. Am.Chem. Soc., 113:6657-6662 (1991). Products from this reaction werepurified via HPLC. Fast atom bombardment mass spectroscopy confirmed themolecular weight of the linear and cyclic peptides. The cyclic disulfidepeptides were negative for free thiol with Ellman's reagent.

The synthetic peptides from Fab 9 were tested for the ability toantagonize α_(v)β₃ and α_(IIb)β₃ and α_(v)β₅ in purified ligand-receptorbinding assays performed as described in Example 5b. The results ofassays with Fab 9 are shown in FIG. 4A. In FIG. 4A, the linear form ofthis peptide, designated Lin-9 and listed in SEQ ID NO 68, was testedfor the ability to block ¹²⁵I-vitronectin binding to α_(v)β₃ (opentriangles), α_(v)β₅ (closed circles) and ¹²⁵I-fibrinogen binding toα_(IIb)β₃ (closed triangles).

Results of competition assays with the Fab 9-derived peptide in SEQ IDNO 68 (FIG. 4A) showed that it completely inhibited the binding ofvitronectin to both α_(v)/β₃ and gpIIb/IIIa reaching maximal inhibitionwith 10⁻⁵ M peptide. No inhibition of binding of vitronectin to α_(v)/β₅was obtained. Thus, the peptide derived from Fab 9 exhibited the sameinhibitory specificity as the antibody itself inhibiting only one typeof vitronectin receptor and gpIIb/IIIa. Similar results were obtainedwith the circularized Fab 9-derived peptide. These results indicate apreferred motif and arrangement of RGD within a peptide in order to bindto and block high affinity ligand binding over that observed with thesimple RGD motif without flanking sequences.

In examining the peptides for activity, two criteria for judging theirefficacy, affinity and specificity were used. The proto-typical RGDmotif found in fibronectin, GRGDSP (SEQ ID NO 66), was used as apositive control in inhibition assays because it antagonized all threeintegrins. The assay results are shown in FIG. 4B. In FIG. 4B, the sameconcentration range of peptide GRGDSP (SEQ ID NO 66) was tested in anidentical assay. All data are expressed as the % of control binding inthe absence of inhibitor. Data are expressed as the average oftriplicate data points. This is representative of four experiments inwhich nearly identical results were obtained in each repetition.

It is important to note that GRGDSP did antagonize α_(v)β₅. In fact, ofthe three integrins tested, α_(v)β₅ had the highest apparent affinityfor this peptide. Interestingly, the heavy chain CDR3 peptide derivedfrom Fab 9 inhibited ligand binding to α_(v)β₃ and α_(IIb)β₃, but didnot antagonize α_(v)β₅ as shown in FIG. 4A. Thus, the linear Fab 9peptide, designated Lin-9, is the first RGD-containing peptide withspecificity for α_(v)β₃ over α_(v)β₅. Thus, the synthetic peptidederived from this CDR3 emulated the activity of the whole syntheticantibody and satisfied the test of maintaining specificity for a preciseintegrin target.

Moreover, the Fab 9-derived peptide exhibited specificity in contrast toinhibition experiments with a GRGDSP peptide (SEQ ID NO 66) as shown inFIG. 4B. The GRGDSP peptide did not exhibit any receptor specificity inthat it inhibited the binding of all high affinity ligands tested totheir respective receptor, vitronectin to both α_(v)/β₃ and α_(v)β₅ andfibrinogen to gpIIb/IIIa. Thus, the Fab 9-derived peptide retainedreceptor specificity only inhibiting the binding to only one vitronectinreceptor, α_(v)/β₃, despite the removal of the flanking regions of theheavy chain variable domain.

Importantly, no significant differences were observed in the efficacy ofthe cyclic and linear peptides in any inhibition studies, indicatingthat cyclization was not a determinant in binding affinity. In addition,no difference was observed between linear peptide forms containingterminal cysteines versus terminal glycines, eliminating the possibilitythat peptide cyclization during the time course of the inhibition assayhad an effect on the efficacy of the peptide.

4) Platelet Aggregation Assays

Platelet aggregation assays were performed as described in U.S. Pat. No.5,114,842. In platelet aggregation assays using the Fab-displayed RGDC2binding site protein labeled Mab 19 as listed below in Example 4B, 6.6uM of the purified soluble RGDC2-expressing Fab inhibited theaggregation of platelets in a time-dependent manner as indicated in adecrease in the amount of light transmitted as compared to control humanFabs. When the concentration of the RGDC2-binding site Fab was decreasedby half, the amount of platelet aggregation increased similarly and thusthe amount of light transmitted increased. When compared insimultaneously performed assays using murine Fabs, in particular with anantibody designated LIBS-1 previously shown to effectively inhibitplatelet aggregation, the RGDC2-binding site Fab of this invention wasequally effective at inhibiting platelet aggregation. Thus, the methodsof this invention allow for the production of novel human Fab moleculesthat express synthetic binding sites such as RGD peptide as well as thecyclized RGD peptide and that exhibit desired therapeuticcharacteristics as demonstrated by the ability of the Mab 19 to inhibitplatelet aggregation.

B. Sequence Determination of the Binding Site Proteins

Nucleic acid sequencing was performed on double-stranded DNA usingSequenase 1.0 (USB, Cleveland, Ohio) encoding the specific solubleFab-displayed synthetic binding site proteins of this inventioncharacterized above. The derived amino acid residue sequences of thespecific Fab-displayed synthetic binding site proteins for receptorsincluding VNR and gpIIb/IIIa were listed earlier in the Section entitled“Binding Site Polypeptides”.

Examination of the sequences revealed homologies within the group andwith other known ligands. The SFG-RGD sequence of Fab 9 (in SEQ ID NO 4from amino acid positions 3 to 8) is found in lamB, in kistrin, and in anumber of other snake venoms. Interestingly, one noted feature is thestrict conservation of RGD-XR (SEQ ID NO 65) which is not found in anyknown ligand. In addition, the resultant derived amino acid residuesequences of both the minimum binding site CDR3 as well as the motifswitched CDR3 monoclonal antibodies of this invention exhibit somedeviations from the expected sequence from the oligonucleotides used inthe PCR reactions for incorporating the binding sites. The minornon-perturbing nucleotide substitutions resulting in the encoding of anon-expected amino acid residue is probably the result of non-fidelityof the PCR amplification.

5. Characterization of Soluble Non-RGD-Containing Motif OptimizedBinding Site Proteins

A. Engineering Antibody Specificity by Motif Optimization and ELISACharacterization

In order to identify non-RGD sequences that could antagonize receptorfunction and to determine whether antibodies could be designed thatdiscriminate between the two β₃-integrins, “motif optimization” wasperformed as described in Example 1D. This involved the construction ofan additional phage library as described in Example 2.

Since Fab 9 as described in Example 4 had the highest affinity of thefirst series of RGD-containing antibodies, the residues corresponding to1-4 and 9-11 of the integrin binding motif in this antibody (as shown inFIG. 1) were maintained in the new phage library. However, residues atpositions 5-8, corresponding to RGDI of Fab 9, were randomized by usingdegenerate oligonucleotide pools (also referred to as oligonucleotidedoping) and PCR as described. The complexity of this library ensured a99% probability that all of the possible amino acid sequences wererepresented in the randomized four residue motif.

This phage library was screened as described in Example 3A forantibodies that could distinguish α_(v)β₃ from α_(IIb)β₃. Two selectionswere performed, one for α_(v)β₃ and one for α_(IIb)β₃. The selectionprocess involved the incubation of the phage library with 20 ug/ml ofcompeting integrin in solution, and subsequent panning on theimmobilized target integrin. For example, to select antibodies withspecificity for α_(IIb)β₃, integrin α_(v)β₃ was incubated with thebacteriophage library in solution prior to panning on immobilizedα_(IIb)β₃. Three rounds of this selection process were performed foreach target receptor and the resulting phage were initially tested forspecificity by ELISA as described in Example 4.

B. Ligand-Receptor Binding Assays and Inhibition of Binding Assays

Antibodies that displayed a preference for the target integrin in ELISAwere screened more rigorously by measuring their ability to antagonizeα_(v)β₃ and α_(IIb)β₃ receptor activity in purified ligand-receptorbinding assays. The method for these binding studies has been describedby Barbas et al., Proc. Natl. Acad. Sci., USA, 90:10003-10007 (1993) andSmith et al., J. Biol. Chem., 265:11008-11013 (1990), the disclosures ofwhich are hereby incorporated by reference.

Briefly, selected purified integrins were separately immobilized inTitertek microtiter wells at a coating concentration of 50 ng per well.After incubation for 18 hr at 4° C., non-specific binding sites on theplate were blocked with 10 mg/ml of bovine serum albumin inTris-buffered saline. For inhibition studies, a range of antibody wastested for the ability to block the binding of ¹²⁵I-vitronectin toα_(v)β₃ and ¹²⁵I-fibrinogen to α_(IIb)β₃. The choice to use twodifferent ligands in comparing the inhibitory activity of antibodies wasa compromise based on a complicated series of circumstances. Ideally thesame ligand would have been used for inhibition studies, but fibrinogendoes not bind α_(v)β₃ when Ca²⁺ is present. In contrast, Ca²⁺ isessential for the rapid binding of fibrinogen to α_(IIb)β₃. Similarcomplications arise when vitronectin is considered because it does notbind identically to the two integrins. Therefore, the optimal ligand wasselected for each integrin. This corresponds to vitronectin for α_(v)β₃and fibrinogen for α_(IIb)β₃. Radiolabeled ligands were used atconcentrations of 1 nM and binding was challenged with unlabeledantibody.

Following a three hour incubation free ligand was removed by washing andbound ligand was detected by gamma counting. The data from this analysiswere highly reproducible with the error between data points typicallybelow 11%. Data are expressed as the average of triplicate data points.All experiments were repeated at least four times.

The ability of two motif optimized Fabs, MTF-2 and MTF-10 to blockligand binding is shown in FIGS. 2A and 2B, respectively. Ligand bindingto α_(v)β₃ as shown by the line marked with the filled-in circles wasmeasured with ¹²⁵I-vitronectin and ligand binding to α_(IIb)β₃ as shownby the line marked with the open circles was assessed with¹²⁵I-fibrinogen. All data are expressed as the % of control binding inthe absence of inhibitor. Non-specific binding was determined bycompetition with RGD peptides and was normally less than 10% of thetotal bound counts.

The IC₅₀ of MTF-2 was 20-fold lower for α_(IIb)β₃ than for α_(v)β₃ (FIG.3A). MTF-10 exhibited a 50-fold greater ability to antagonize α_(IIb)β₃(FIG. 3B). In particular, the MTF-10 Fab that contained the new bindingamino acid motif, GKGDN, as shown in SEQ ID NO 25 from position 6 to 10,at a concentration of 1×10⁻⁸ M maximally inhibited the binding ofvitronectin to IIb/IIIa. In addition, MTF-10 inhibited 60% of thebinding of vitronectin to α_(v)/β₃ when the antibody was used at aconcentration of approximately 1×10⁻⁷ M.

In the same assay NTF-32 displayed a 15-fold preference for α_(IIb)β₃.All of the MTF antibodies exhibited a higher IC₅₀ than the parent Fab 9,indicating that by gaining specificity for α_(IIb)β₃, some affinity forthe integrin was sacrificed.

Thus, by altering a RGD motif by the motif switching methods of thisinvention, antibodies lacking RGD able to exhibit specificity tointegrin receptors were shown to bind high affinity ligands throughRGD-mediated binding interaction.

Interestingly, with this motif optimization no antibodies wereidentified that displayed dramatic binding specificity for α_(v)β₃. Thisindicates that within the context of the Fab 9 scaffold, it is unlikelythat the adhesion motifs can be manipulated to gain substantialspecificity for α_(v)β₃ over α_(IIb)β₃. This is not surprising becauseRGD-based ligands that bind to α_(v)β₃ are not known and do not bindα_(IIb)β₃.

C. Amino Acid Sequence Determination

Nucleic acid sequencing was performed on double-stranded DNA usingSequenase 1.0 (USB, Cleveland, Ohio) encoding the specific solublenon-RGD Fab-displayed motif optimized proteins of this inventioncharacterized above. The derived amino acid residue sequences of thesespecific Fab-displayed proteins for receptors including VNR andgpIIb/IIIa were listed earlier in the Section entitled “Binding SitePolypeptides”.

While the RGD-engineered CDR3 monoclonal antibodies all contained RGDwithin the CDR3 at Kabat positions 100 a-c at the placement of the apexof the extended hair-pin loop of the CDR3 heavy chain, the motifoptimized sequences exhibited a number of amino acid substitutions inthe RGDI (SEQ NO ID 53) mutated region of Fab 9. The CDR3 listed in SEQID NO 4 was re-selected following motif switching mutagenesis one timeby screening on gpIIb/IIIa and three times by screening on the VnRreceptor, α_(v)/β₃.

Thus, this RGD-containing CDR3 amino acid residue sequence represents amore common member of the library that binds to both gpIIb/IIIa and theβ3-containing VnR receptor but not to the β5-containing VnR receptor.

Four motif optimized antibodies were obtained that bound preferentiallyto α_(IIb)β₃ (gpIIb/IIIa).

These are termed MTF-2, MTF-10, MTF-32 and MTF-40 respectivelydesignated MTFIIb/IIIa-2 (SEQ ID NO 26), MTFIIb/IIIa-10 (SEQ ID NO 25),MTFIIb/IIIa-32 (SEQ ID No 32) and MTFIIb/IIIa-40 (SEQ ID NO 34) aslisted herein with their corresponding SEQ ID NOs. The amino acidresidue sequences as listed contain the heavy chain CDR3 bordered byframework 3 on the amino terminus and framework 4 on the carboxyterminus as shown in FIG. 1.

None of these MTF antibodies contained the RGD motif, although in threeof four cases the R and D were conserved. No functional substitutionsfor D were observed in any of the selected clones. In MTF-10, thearginine was replaced by lysine, a substitution that has been found inother ligands with specificity for α_(IIb)β₃, including the snake venombarbourin. Interestingly, the binding pocket of β₃-integrins canapparently tolerate rather dramatic substitutions at the “G” positionbecause residues which have substantial side chains were accepted inthis position. MTF's 2, 10 and 32 had lower IC₅₀'s for α_(IIb)β₃ thanα_(v)β₃ in purified ligand receptor binding assays.

D. Affinity Measurement and Analysis

A comparison of absolute antibody affinities was then performed withsurface plasmon resonance, which is a highly accurate means of assessingligand affinities in real time. See, Altshuh et al., Biochem.,31:6298-6304 (1992); Chaiken et al., Anal. Biochem., 201:197-210 (1992);Eddowes, Biosensors, 3:1-15 (1987); Fagerstam et al., J. Chromatoar.,597:397-410 (1992); Johnsson et al., Anal. Biochem., 198:268-277 (1991);Karlsson et al., J. Immunol. Methods, 145:229-240 (1991); and VanCott etal., J. Immunol. Methods, 146:163-176 (1992), the disclosures of whichare hereby incorporated by reference. SPR is a phenomena that occurswhen incoming photons interact with electrons in a biosensor chip. SPRcan be used to detect binding between a reactant immobilized on a goldchip and an interacting molecule in solution. The reflective index ofthe biosensor chip changes as a result of the binding between ligand insolution and the immobilized receptor. This technology has been adaptedto study ligand binding to integrins.

SPR was performed using the BIAcore plasmon resonance instrument fromPharmacia (Pharmacia LKB, Piscataway, N.J.). Integrins were immobilizedon the biosensor chip with N-hydroxysuccininimide andN-ethyl-N′-(3-diethylaminopropyl)carbodiimide according to the methodsoutlined by Pharmacia. The sensor surface was first activated with NHSand EDC. Integrins were coupled by injecting 30 uL of a 100 ug/ml sampleof purified integrin onto the sensor chip surface. Ethanolamine was usedto block unreacted moieties on the sensor chip surface.

Initial studies were done to measure vitronectin binding to α_(v)β₃ andfibrinogen binding to α_(IIb)β₃. Both binding phenomena were inhibitedby RGD peptides and were divalent metal ion-dependent. In fact, theassociation rate constants measured with plasmon resonance werecomparable to that which was recently reported with radioligand bindingstudies. For example, the association rate constant for fibrinogenbinding to α_(IIb)β₃ was 8.5×10⁵ M⁻¹s⁻¹ using solid-phase radioligandbinding assays, and 6×10⁵M⁻¹s⁻¹ using SPR. The fibrinogen bound toα_(IIb)β₃ on the BIAcore sensor chip also exhibited transition to anon-dissociable state. Between 60 and 70% of the bound fibrinogen didnot freely dissociate and had to be removed with more stringentregeneration conditions. All of these data validate plasmon resonance asa means of measuring ligand binding to integrins.

The association and dissociation rate constants (k₁ and k⁻¹) forsemi-synthetic antibodies were obtained from BIAcore measurements asfollows. To derive k⁻¹, a pulse containing ligand was passed through thesensor chip. At the end of the association phase the flow was changed tobuffer without ligand and the change in response unit (RU) was measuredas a function of time. The dissociation rate constant k⁻¹ is derivedfrom equation 1.k ⁻¹=(ln RU ₀ /RU _(t))/t−t ₀  Equation 1RU₀ is the initial response unit due to binding of antibody and RU_(t)is the response unit remaining following dissociation. Time isdesignated as t.

To obtain the association rate constant k₁, an antibody was passedthrough the sensor chip containing unoccupied integrin and the responseunit as a result of binding is again measured as a function of time. Theassociation rate constant, k₁, is calculated as shown in equation 2.k ₁=[((dRU/dt)/RU)−k ⁻¹ ]/L  Equation 2L is the concentration of ligand. Measurements of dRU/dt are obtained atseveral ligand concentrations. The overall k_(D) of each binding eventis derived by simple division shown in equation 3.k _(D) =k ⁻¹ /k ₁  Equation 3Duplicate measurements of k⁻¹ yielded identical values in all cases. Inthe case of k₁, the error was typically less than 13% across severalligand concentrations.

The results of the surface plasmon resonance affinity analysis forα_(v)β₃ and α_(IIb)β₃ are shown in Table 1. The association (k₁ ork_(on)) and dissociation (k⁻¹ or k_(off)) rate constants betweensynthetic antibodies and the two β₃-integrins was determined in realtime with surface plasmon resonance. Data collection and analysis wereperformed with purified integrins and Fab fragments as described herein.Measurements were made in either 2 mM Ca²⁺ or 0.2 mM Mn²⁺, both of whichhave been found to saturate the ligand binding response of theβ₃-integrins.

TABLE 1 Anti- Cation Receptor body K_(on) (M⁻¹s⁻¹) K_(off) (s⁻¹) K_(d)(M) Fab-9 1.3 × 10⁵ 7.0 × 10⁻⁴ 5.0 × 10⁻⁹ α_(IIb)β₃ MTF-2 7.8 × 10⁴ 1.3× 10⁻³ 1.7 × 10⁻⁸ MTF-10 1.7 × 10⁵ 1.2 × 10⁻³ 7.0 × 10⁻⁹ MTF-32 6.8 ×10⁵ 2.0 × 10⁻³ 2.9 × 10⁻⁹ MTF-40 7.7 × 10⁵ 8.4 × 10⁻⁴ 1.1 × 10⁻⁹ Ca²⁺Fab-9 1.4 × 10⁶ 2.3 × 10⁻³ 1.6 × 10⁻⁹ (2 mM) α_(v)β₃ MTF-2 1.4 × 10⁴ 1.8× 10⁻³ 1.3 × 10⁻⁷ MTF-10 3.4 × 10³ 1.7 × 10⁻³ 5.0 × 10⁻⁷ MTF-32 1.5 ×10⁵ 1.0 × 10⁻³ 6.7 × 10⁻⁹ MTF-40 1.0 × 10⁵ 1.1 × 10⁻³ 1.1 × 10⁻⁸ Fab-94.0 × 10³ 6.6 × 10⁻⁴ 1.6 × 10⁻⁷ α_(IIb)β₃ MTF-2 1.8 × 10⁴ 1.7 × 10⁻⁴ 1.0× 10⁻⁸ MTF-10 4.3 × 10⁴ 3.6 × 10⁻⁴ 8.3 × 10⁻⁹ MTF-32 1.1 × 10⁵ 1.2 ×10⁻⁴ 1.0 × 10⁻⁹ MTF-40 4.1 × 10⁴ 1.5 × 10⁻⁴ 3.6 × 10⁻⁹ Mn²⁺ Fab-9 2.4 ×10⁵ 6.5 × 10⁻⁵  2.8 × 10⁻¹⁰ (0.2 mM) α_(v)β₃ MTF-2 3.8 × 10⁴ 1.0 × 10⁻³2.6 × 10⁻⁸ MTF-10 3.6 × 10⁴ 1.8 × 10⁻³ 5.0 × 10⁻⁸ MTF-32 1.3 × 10⁵ 8.7 ×10⁻⁴ 6.7 × 10⁻⁹ MTF-40 1.1 × 10⁵ 9.6 × 10⁻⁴ 8.8 × 10⁻⁹

These measurements demonstrated that the affinities of the MTFantibodies are higher for α_(IIb)β₃ than α_(v)β₃. MTF-10 displayed thehighest level of specificity, as its affinity for α_(IIb)β₃ was 100-foldgreater than for α_(v)β₃ (Table 1).

One high-affinity antibody, MTF-40, survived the selection process forα_(IIb)β₃, but did not display dramatic specificity in the purifiedligand-receptor binding assays. The sequence of the binding motif inthis antibody is RNDS (SEQ ID NO 34 from position 7-10), another non-RGDsequence. Plasmon resonance analysis of the affinity for this Fab showedthat in Ca²⁺, it had a 10-fold higher affinity for α_(IIb)β₃ thanα_(v)β₃. Importantly, in physiologic [Ca²⁺], this antibody alsodisplayed the highest k_(d) for α_(IIb)β₃ of any of the antibodies asmeasured by surface plasmon resonance (1.1×10⁻⁹M). It is likely that thepreference of this antibody for α_(IIb)β₃ was not detected in theinhibition assays because it retained very high affinity for α_(v)β₃.Because the association rate of the native ligands is so slow, theligand binding assay is not sensitive enough to detect small differencesin binding affinity if the affinity of the antibody is very high. Asimilar scenario is evident for the parent Fab 9, which has an obviouspreference for α_(v)β₃ when binding is measured directly with plasmonresonance (Table 1), yet this antibody blocked ligand binding to bothintegrins identically as described by Barbas et al., Proc. Natl. Acad.Sci. USA, 90:10003-10007 (1993).

1) The Binding of Recombinant Fab's to Integrins is Dissociable.

It is well established that the ligands for β₃-integrins bind in anon-dissociable manner. This has been observed in purified systems andon whole cells. See, Muller et al., J. Biol. Chem., 266:3579-3585 (1991)and Orlando et al., J. Biol. Chem., 266:19543-19550 (1991), thedisclosures of which are hereby incorporated by reference. However,ligand mimicking peptides with the RGD sequence freely dissociate fromthese receptors. Consequently, it was important to determine how thesemi-synthetic RGD-containing and non-RGD-containing motif optimizedantibodies bound these integrins.

The binding of Fab 9 is shown as an example in this analysis, but itsbinding behavior was essentially identical to that of the MTFantibodies. The ability of ¹²⁵I-Fab 9 to dissociate from purifiedα_(v)β₃ was compared to that of ¹²⁵I-vitronectin in purifiedligand-receptor binding assays performed as described in Example 5B.Both ligands were incubated with purified α_(v)β₃ for four hours. Thenbuffer containing radioligands was replaced with binding buffer lackingligand to allow dissociation. An effort was also made to promotedissociation by adding 10 uM GRGDSP (SEQ ID NO 66) after the bindingstep. This concentration of RGD peptide is 100-fold above the IC₅₀ ofthis peptide when it is simultaneously added with ¹²⁵I-vitronectin andα_(v)β₃. Following a two hour incubation to allow dissociation, bufferwas removed and the amount of ¹²⁵I-ligand remaining bound to integrinwas measured. The remaining bound ligand was compared to the amount ofligand bound at the beginning of the dissociation step.

From these assays, it was determined that 90% of the bound Fab 9 freelydissociated from α_(v)β₃. No competing RGD peptide was necessary topromote dissociation. In contrast greater than 65% of the boundvitronectin remained associated with α_(v)β₃. Competition with RGDpeptide after the binding event had little effect on dissociation.Similar data were obtained for α_(IIb)β₃ and also for the othersynthetic antibodies. Unlike the native adhesive ligands for theβ₃-integrins, the synthetic antibodies against the ligand binding sitefreely dissociated from integrin.

2) Measuring Ligand Association and Dissociation Constants withSynthetic Antibodies

The dissociable nature of antibody binding to the ligand binding pocketallowed for the first measurement of molecular association anddissociation rates for this site. The association rates of naturalligands for both β₃-integrins have been previously established, but dataregarding dissociation was not obtained because of the stabilizedbinding between adhesive proteins and integrins.

To accurately quantify association and dissociation rates between thesemi-synthetic Fab antibodies of this invention and the β₃-integrins,surface plasmon resonance was as described above. To validate thisapproach for integrins, preliminary binding studies were done betweenfibrinogen and α_(IIb)β₃ and between vitronectin and α_(v)β₃ asdescribed above.

The binding of the MTF antibodies and Fab 9 were compared on bothα_(v)β₃ and α_(IIb)β₃. The association and dissociation constants andthe k_(D)'s of each antibody for both integrins are listed in Table 1shown above. In prior studies it was determined that ligand binding toeither integrin could be maximally supported by either 2 mM Ca²⁺ or 200uM Mn²⁺, and that these ions had different effects on ligand association(Smith et al., J. Biol. Chem., 266:11429-11432 (1991). Consequently, theassociation and dissociation rate constants for antibodies weredetermined under both conditions. Because the MTF antibodies lacked RGD,these data conclusively show that absolute adherence to this motif isnot necessary for high-affinity binding to β₃-integrins. Even though theMTF antibodies all showed substantial preference for α_(IIb)β₃, they didhave reasonable affinity for α_(v)β₃. In fact, the association rateconstants of the MTF antibodies for α_(v)β₃ are between 10³ and10⁵M⁻¹s⁻¹, a range identical to that found for the native RGD containingligands. Therefore, if presented in the proper context, proteinsequences other than RGD can bind both β₃-integrins.

3) Divalent Ions Regulate k₁ for Synthetic Antibodies

Divalent cations regulate the association of natural ligands with bothα_(v)β₃ and α_(IIb)β₃. The binding of the synthetic antibodies againstthe ligand binding site were also markedly influenced by the type ofdivalent ion present. Since these antibodies freely dissociated fromintegrin they provided the first opportunity to measure the influence ofdivalent metals on both association and dissociation.

To provide an easy means of examining the influence of differentdivalent ions on antibody binding, the ratio of k₁ (k_(on)) in Mn²⁺ tok₁ in Ca²⁺ was calculated. The same calculation was performed for k⁻¹(k_(off)). The results are shown in Table 2. The values used for thesecalculations were derived from the affinity measurements with plasmonresonance which are listed in Table 1. Smith et al., J. Biol. Chem.,265:11008-11013 (1990) found that integrin α_(IIb)β₃ always boundligands poorly in buffer containing only Mn²⁺, consequently the ratio'sof k₁ for this integrin are always much lower than 1. Ratios of oneindicate equivalent association in both ions. Ratios above one indicatefaster association in Mn²⁺.

TABLE 2 α_(v)β₃ α_(v)β₃ α_(IIb)β₃ α_(IIb)β₃ Synthetic k_(on) k_(off)k_(on) k_(off) Antibody Mn/Ca Mn/Ca Mn/Ca Mn/Ca Fab-9 0.17 0.02 0.030.94 MTF-2 2.71 0.55 0.23 0.13 MTF-10 10.5 1.05 0.25 0.30 MTF-32 0.860.87 0.16 0.06 MTF-40 1.10 0.87 0.05 0.17

As shown in Table 2, there was a wide variation in the effect ofdivalent ions on association rate constants. The parent antibody, Fab 9,had a substantially faster on-rate for a in Ca²⁺ than in Mn²⁺.Conversion of the RGDI (SEQ ID NO 4 from position 7-10) sequence of Fab9 to the RTDQ (SEQ ID NO 26 positions 7-10) sequence in MTF-2 completelyswitched the cation preference for binding to Mn²⁺. The antibody withthe largest difference in association rate constants was MTF-10, whichhas a KGDN (SEQ ID NO 25 from position 7-10) sequence. The associationrate constant for this antibody was 10-fold higher in Mn²⁺ than in Ca²⁺.This was a drastic reversal of the ion preference shown by the parentantibody, indicating that sequence changes within the four core residuesof the integrin recognition motif can dramatically affect a ligand'sbehavior with respect to divalent ions.

Integrin α_(IIb)β₃ binds all of its natural ligands faster in Ca²⁺ thanin Mn²⁺. This trend is also evident with the semi-syntheticRGD-containing and non-RGD-containing motif optimized antibodies of thisinvention. Regardless of the antibody tested, the ratio of associationrate constants in Mn²⁺ versus Ca²⁺ is always substantially below 1.However, sequence changes in residues 5-8 can alter this ratio as muchas five-fold. MTF-40 has a Mn²⁺/Ca²⁺ preference of 0.05, but for NTF-2the ratio is 0.23. The dissociation rate constants also varied with thetype of divalent ion present, but there was no obvious correlationbetween the influence of ions on k₁ and k⁻¹ for a given antibody. Forexample, when binding α_(v)β₃ MTF-32 had nearly identical ratios for k₁and k⁻¹, 0.86 and 0.87. In contrast, the ratios for MTF-10 on α_(v)β₃were 10 and 1. Consequently, divalent ions can influence the ligandon-rate without necessarily influencing its off-rate.

These results indicate that prior to the transition to a stable complexbetween integrins and their natural ligands, ligand association anddissociation can be regulated independently. Depending on the timerequired for transition to a stable complex, the independent regulationof binding and dissociation could be important for regulating cellularextension on the extracellular matrix.

4) Antibody Association Rate Constants Validate the Antibody SelectionTechnique

Panning as described in Example 2B is becoming a standard technique foridentifying high affinity binders from within phage libraries, yetlittle systematic study has been reported regarding the efficiency ofthe panning approach. The scheme for selecting specific antibodiesagainst α_(IIb)β₃ involved competing with α_(v)β₃ in solution andpanning the non-bound antibodies on immobilized α_(IIb)β₃. The panningstep was set at two hours, and the non-bound phage were removed with aone hour wash period.

To assess the success of the competitive selection process designed foridentifying antibodies with specificity for α_(IIb)β₃, the ratio of theassociation rate constant for α_(v)β₃ to the association rate constantfor α_(IIb)β₃ was calculated. The values for this derivation wereobtained from plasmon resonance measurements listed in Table 2. Ratio'sof 1 indicate the antibody has an identical ability to associate withboth receptors. Ratio's of less than 1 indicate a preference forα_(IIbβ) ₃. All antibodies from the MTF series have lower ratio's thanthe parent antibody, Fab 9.

TABLE 3 k_(on) in Ca²⁺ k_(on) in Mn²⁺ Antibody α_(v)β₃/α_(IIb)β₃α_(v)β₃/α_(IIb)β₃ Fab-9 10.7 60 MTF-2 0.17 2.1 MTF-10 0.02 0.83 MTF-320.22 1.1 MTF-40 0.12 2.6

This comparison reveals that the motif optimization and selection washighly successful. The parent antibody, Fab 9, displayed on-rates thatfavored α_(v)β₃. In buffer containing Ca²⁺, the k₁ ratio for thisantibody was 10, indicating preference for α_(v)β₃. All of the MTFantibodies, which were selected on α_(IIb)β₃, had ratios of less than 1,indicating a preference for α_(IIb)β₃. Although the absolute values ofratios of k₁, were different in Mn²⁺, the same trend was evident.Inspection of the overall k_(D)'s for the MTF antibodies (Table 1) showsthat the overall antibody binding affinity was also generally higher forα_(IIb)β₃ than α_(v)β₃. These results indicate that the competitiveselection process properly identified antibodies with specificity forα_(IIb)β₃.

E. Comparison of Fab 9 and MTF-10 in Platelet Aggregation and CellAdhesion

All of the initial selection and characterization of the semi-syntheticFab antibodies of this invention relied largely upon the use of purifiedintegrins. Since the MTF-antibodies blocked the function of α_(IIb)β₃and have potential therapeutic use in the prevention of thrombosis, theability of recombinant Fabs to block platelet aggregation was measuredas described in Example 4. Washed platelets (1×10⁸) were placed inTyrode's buffer containing 2 mM Ca²⁺ and 100 ug/ml of fibrinogen andaggregation was stimulated by addition of 20 uM ADP. Aggregation wasmeasured as light transmission through the platelet suspension using aScienceo aggregometer.

As described in Example 4A4), Fab 9 blocked one-half of the aggregationat a concentration of near 5 nM. This antibody completely blockedaggregation at an antibody concentration of 20 nM. Only slightly moreMTF-10 was required for inhibiting aggregation, with half-maximalinhibition at 20 nM Fab and complete inhibition at 100 nM. Theaggregation data were also in accordance with the affinities of thesetwo antibodies as measured by plasmon resonance. The other MTFantibodies were similar to MTF-10 in their ability to interfere withplatelet aggregation. These findings show that the semi-syntheticantibodies of this invention can abrogate platelet aggregation andsubstantiate their potential as anti-thrombotic agents.

Analysis of MTF-10 in purified systems indicated that it should have asubstantially reduced ability to bind α_(v)β₃ on the cell surface. Thisassay, performed as described in Example 4, was performed with wholecell adhesion assays using M21 melanoma cells because they adhere toRGD-ligands primarily via α_(v)β₃. M21 cells were allowed to adhere to aconcentration range from 1 to 10 ug/ml of immobilized Fab 9 and MTF-10.

The cells were able to adhere to the RGD-containing Fab, but were unableto bind to MTF-10 which has a KGDN (SEQ ID NO 25 from position 7-10)sequence instead of RGDI (SEQ ID NO 4 from position 7-10). AntibodyMTF-10 did support the adhesion of chinese hamster ovary cellsexpressing recombinant human α_(IIb)β₃.

F. Preparation of Synthetic Peptides Derived from Fab Antibody HCDR3Protein Sequences

The ability of peptides derived from MTF-32 and MTF-40 to block ligandbinding to the two β₃-integrins was measured. Peptides derived from thesequence of MTF-10, which has a KGD sequence, were not tested becausepeptides containing this motif have previously been shown bind andinhibit α_(IIb)β₃. See Ginsberg et al., J. Biol, Chem., 260:3931-3936(1985).

Since antibodies MTF-32 and MTF-40 had a higher affinity for α_(IIb)β₃in plasmon resonance analysis (Table 1), and because these antibodieslacked RGD, peptides derived from these CDR's were also tested for theirability to block ligand binding to α_(v)β₃ and α_(IIb)β₃. Both cyclicand linear versions of peptides derived from MTF-32 and MTF-40 weresynthesized as described for Fab 9 in Example 4A3). The data presentedherein is analyzed in view of the results of similar peptide competitionassays with Fab 9 as described in Example 4A3).

In FIG. 4C, peptides derived from HCDR3 of MTF-40, peptide CSFGRNDSRNC(SEQ ID NO 69) and peptide GCSFGRNDSRNCY (SEQ ID NO 70) weresynthesized. The cyclic form is indicated in the figure with opensquares and open triangles while the linear form is shown as closedcircles and closed triangles. The ability of these peptides to blockligand binding to α_(v)β₃ (closed triangles and open squares) andα_(IIb)β₃ (closed circles and open triangles) was compared. All data areexpressed as the percent of control binding in the absence of inhibitor.Data points are the average of triplicate values. This experiment isrepresentative of three repetitions in which nearly identical resultswere obtained.

In FIG. 4D, similar measurements were made for peptides derived fromHCDR3 of NTF-32, circular peptide CSFGRTDQRNC (SEQ ID NO 71) and linearpeptide GCSFGRTDQRNCY (SEQ ID NO 72). Linear (closed triangles andclosed circles) and cyclic (open triangles and open squares) peptideswere tested for the ability to block ligand binding to α_(v)β₃ (closedtriangles and open squares) and α_(IIb)β₃ (closed circles and opentriangles). Again the data are the average of triplicate points and theexperiment is representative of three repetitions with similar results.

Both linear and circular peptides 32 and 40 blocked fibrinogen bindingto α_(IIb)β₃, but did not interfere with ligand binding to α_(v)β₃ overthe range of peptide concentrations tested (from 1×10⁻⁴ to 1×10⁻¹¹ M) asshown in FIGS. 4C and 4D. Higher concentrations of peptide were notevaluated because several non-specific peptides began to disrupt ligandbinding in this assay at concentrations of 5×10⁻⁴ M. For integrinα_(IIb)β₃, the IC₅₀ of peptide 40 is 5×10⁻⁷M and that of peptide 32 is2+10⁻⁶M. The activity of the peptides was not enhanced by cyclization.Although the affinity of these peptides for α_(IIb)β₃ is low compared toGRGDSP (SEQ ID NO 66) (FIG. 4B), they do maintain the bindingspecificity exhibited by the parent antibodies.

G. Summary

With the methods of this invention, integrin ligands, and antagonists,that maintain a high degree of specificity were designed. Semi-syntheticFab antibodies were re-designed as integrin ligands by manipulating theantigen binding site. The placement of an integrin ligand motif into thecomplementarity determining region of a human antibody's heavy chain,and optimization of this motif by selecting high affinity antibodies wasaccomplished with the phage-display libraries. Interestingly, importantdifferences were observed in the way that the synthetic antibodies andnative ligands bind to integrins. Native adhesive ligands for theintegrins typically have very low association rate constants, butultimately bind in a non-dissociable manner. In contrast, this inventionhas provided synthetic antibodies that bound the β₃ integrins rapidly,with at least one interaction of greater than 1×10⁶ M⁻¹s⁻¹. This rate is600-fold faster than the association of vitronectin for α_(v)β₃.However, unlike vitronectin, all of the antibody ligands bound in acompletely dissociable manner. The inability of the semi-syntheticantibodies to bind tightly, i.e., in a non-dissociable manner, indicatesthat the native ligands have ancillary contact surfaces aside from RGDthat contribute to the stabilized binding state. These hypothesizedsecondary contact points are also a likely means of obtaining anotherlevel of specificity between integrins and their natural ligands.

In most cases, therapeutic application of antagonists of β₃-integrinswill require some measure of specificity for either α_(v)β₃ orα_(IIb)β₃. Semi-synthetic antibodies that could distinguish these twointegrins were prepared with the methods of this invention and thus themethods provide for affinity enhancement for α_(IIb)β₃. Three of theantibodies in the motif optimized (MTF) series showed preference forα_(IIb)β₃. MTF-10 showed a 100-fold higher k_(D) for α_(IIb)β₃ thanα_(IIb)β₃.

Interestingly, the same selection strategy to find antibodies withpreference for α_(v)β₃ was used but without success. Fab 9, which wasoriginally identified by panning on pure α_(v)β₃, did show preferencefor this integrin in plasmon resonance binding studies, antibodies withgreater specificity were not obtained by optimizing the motif in Fab 9.The complexity of the library screened was sufficient to ensure that allpotential amino acid sequences were present in the motif optimization,leading to the conclusion that the within the context of the adhesionmotif shown in FIG. 1, the set of all sequences that bound α_(v)β₃ alsobound α_(IIb)β₃. Interestingly, no natural ligands have been identifiedthat bind α_(v)β₃ substantially better than α_(IIb)β₃.

Although peptidomimetics and cyclic peptides have been reported to havea high degree of specificity for α_(IIb)β₃ as described by Isoai et al.,Cancer Lett., 65:259-264 (1992), compounds that have good preference forα_(v)β₃ have not been identified. Some snake venoms have also beenreported with preference for α_(v)β₃, but the best example, cerastin,had only a 20-fold lower IC₅₀ for α_(v)β₃ than α_(IIb)β₃. Collectively,these data indicate that it will be substantially more challenging toobtain an RGD-based antagonist with a large degree of selection forα_(v)β₃.

Since all of the potential natural amino acid sequences with the MTFselection strategy has been exhausted with the methods of thisinvention, it is likely that α_(v)β₃-specific antagonists will requireaccess to functional moieties not present in the 20 amino acids, andwill have to be organically synthesized. This does not exclude thepossibility that antibodies can be further engineered to gain selectionfor α_(v)β₃. For example, the HCDR3 of Fab 9 can be recombined withlibraries of light chains in order to build in other contact sitesspecific for α_(v)β₃. Alternatively, five other CDR's are present in theantibody and could be genetically manipulated to achieve the same end.

The integrin ligand binding pocket could also accommodate more proteinsthan originally thought. The RGD motif is certainly not an absoluterequirement for occupation of the ligand binding site of theβ₃-integrins. By starting with the optimal presentation of the ligandmotif in Fab 9 (FIG. 1), and then randomizing the RGDX positions,numerous non-RGD sequences were identified that bound and antagonizedboth β₃-integrins. None of these sequences were observed by theselection of linear or disulfide constrained phage libraries onintegrins as described by O'Neil et al., Proteins, 14:509-515 (1992).Such libraries provided only the previously observed RGD and KGD motifs.In fact, in addition to the MTF series of antibodies, four otherantibodies lacking RGD that bound both α_(v)β₃ and α_(IIb)β₃ wereidentified. Three of these antibodies displayed IC₅₀ values 20 to50-fold higher than Fab 9 in ligand binding in purified assays. The CDRsequences of these antibodies at positions 5-8 (FIG. 1) were RWDA (SEQID NO 73), RADR (SEQ ID NO 74) and KRDM (SEQ ID NO 75). One antibodywith sequence RVDD (SEQ ID NO 76) had an IC₅₀ value comparable to thatof Fab 9 but did not distinguish the two β₃-integrins.

In conjunction with the sequences of the MTF antibodies as shown inSection B, these data indicate that adhesion motif position #6 (FIG. 1)which corresponds to the “G” position in the RGD, is highly permissive.G was found to be substituted by A,D,N,R,Q,S,T and W. A full stearicspectrum of side chains was tolerated in this position. Importantly, theantibodies lacking RGD had association rates that rivaled the fastestassociation partner for a β₃-integrin, fibrinogen. Therefore, thepotential ligands for the β₃-integrins should be expanded to includeproteins with RXD motifs. It is possible that further divergence fromthe RGD sequence at other positions could also be tolerated becauseMTF-10, which has a KGD at positions 5-7 (FIG. 1), still bound α_(v)β₃at a rate comparable to vitronectin, the natural ligand for thisintegrin. Extending the definition of β₃ ligands to sequences of (K/R)XDshould be considered. This would vastly increase the number of potentialligands for integrins. This is illustrated by a computer search of theProtein Identification Resource Database using the RTDQ (SEQ ID NO 32from position 7-10) sequence of antibody MTF-32. Of 56,849 sequences inthe database, 3,026 contained RTDQ. Similar numbers were obtained forRNDS (SEQ ID NO 34 from position 7-10), the active sequence withinMTF-40. Consequently, the pool of potential β₃ ligands is much largerthan originally believed.

The sequences we identified at motif positions 5-8 were also distinctfrom two antibodies raised by immunization that bind the ligand bindingsite of α_(IIb)β₃ as described by Taub et al., J. Biol. Chem.,264:259-265 (1989) and Tomiyama et al., Blood, 79:2302-2312 (1992), thedisclosures of which are hereby incorporated by reference. These areOPG2 and PAC-1, both of which have an RYD motif in CDR3. The methodsused herein did not result in any antibodies with this sequence. It islikely that PAC-1 and OPG2 which have reported high specificity forα_(IIb)β₃ also derive binding affinity from secondary contacts withintegrin that are contributed by other CDR's in the antibody.

Much recent effort has been placed on screening for ligand motifs usingrandom peptide libraries in conjunction with phage-display technology asreferred to in the Background. The methods of this invention differedsubstantially in that the CDR of an antibody was used as a vehicle forpresentation of the peptide motif. Therefore, whether synthetic peptidesderived from the selected sequences would behave faithfully as highfidelity antagonists was not known. In fact, the HCDR3-derived peptidesof this invention did block integrin function. More importantly though,these peptides mimicked the target specificity of the parent antibody.Even though both β₃-integrins and the highly related integrin α_(v)β₃bound to the proto-typical RGD sequence, GRGDSP (SEQ ID NO 66), the RGDpeptide derived from Fab 9 bound only the β₃-integrins. Similar targetspecificity was observed for peptides derived from MTF-32 and MTF-40.Like the parent antibodies, peptides 32 and 40 showed specificity forα_(IIb)β₃ over α_(v)β₃. It is unclear whether the selection of theproper flanking residues in the antibody CDR contribute more contactswith integrin, or whether these flanking residues have conformationallyconstrained the angle that defines the orientation of the side chains ofthe R and D, a parameter known to influence receptor binding affinity.Since the cyclic peptides provided no advantage over the linear peptidesequences in terms of affinity or selectivity, the optimal flankingresidues may add additional contact points for the integrin. It is alsoreasonable to assume that the antibody scaffold with its interactingloops displays the HCDR3 in a manner which is not easily mimicked bysimple cyclization.

Thus, this invention demonstrates the potential for using CDR's as adesign template for obtaining inhibitory CDR-derived peptides. Thefindings presented here indicate that antigen binding peptides derivedfrom the antibody's CDR sequence could be optimized with phage-display.

A major advantage to building human antibodies that bind the integrinligand binding site is that they could have immense therapeuticapplication, particularly because they should circumvent a host immuneresponse. The two β₃ integrins have been implicated in numerousdiseases. An antagonist of the platelet integrin would arrest plateletaggregation, and could find wide application in treating thromboticepisodes. In fact, antagonists of α_(IIb)β₃ have been effective atblocking platelet function in vivo. The findings presented herecertainly show that a semi-synthetic approach to generatinganti-integrin antibodies is valuable. MTF-10, which has substantiallyhigher affinity for α_(IIb)β₃ over α_(v)β₃ (100-fold) is a goodcandidate for testing anti-platelet activity in in vivo models ofthrombosis. The fact that some of the semi-synthetic antibodiesantagonized both β₃-integrins with high affinity may be extremely usefulin combating certain types of metastasis. It is now thought thatmelanoma is often carried through the vasculature by adhesion tocirculating platelets. This cell-cell bridge is thought to involveα_(v)β₃ on the tumor cell and α_(IIb)β₃ on the platelet. Blocking bothends of this adhesion event with the semi-synthetic antibodies describedhere would seem to present a superb therapeutic strategy.

6. Preparation of Synthetic Binding Sites Within the Light Chain CDR3Domain of a Phagemid Fab Display Protein Produced by a DicistronicExpression Vector

In addition to creating synthetic binding sites within the heavy chainof CDR3,in a phagemid-expressed Fab, similar sites were produced in thelight chain of CDR3. For this aspect of this invention, the phagemidexpression vector, pC3AP313, containing heavy and light chain sequencesfor encoding a human antibody that immunoreacted with tetanus toxin, wasused as a template for PCR. The vector was prepared as described inExample 1 while the PCR protocol was performed as described below usingoverlap PCR.

For overlap PCR, each set of PCR reactions were performed in a 100 ulreaction containing 1 ug of each of oligonucleotide primers listed belowin a particular pairing, 8 ul 2.5 mM dNTP's (dATP, dCTP, dGTP, dTTP), 1ul TAQ POLYMERASE, 10 ng of template p7EIII, and 10 ul of 10× PCR bufferpurchased commercially (Promega Biotech). The amplification cycles wereperformed as described in Example 1A. The resultant PCR amplificationproducts were then gel purified as described in Example 1A and used inan overlap extension PCR reaction with the products of the second PCRreaction, both as described below, to recombine the two products intoreconstructed heavy chains containing mutagenized CDR3.

To amplify the 5′ end of the light chain from framework 1 to the end offramework 3 of pC3AP313, the following primer pairs were used. The 5′coding oligonucleotide primer, KEF, having the nucleotide sequence 5′GAATTCTAAACTAGCTAGTCG3′ (SEQ ID NO 59), hybridized to the noncodingstrand of the light chain corresponding to the region 5′ of andincluding the beginning of framework 1. The 3′ noncoding oligonucleotideprimer, KV12B, having the nucleotide sequence 5′ ATACTGCTGACAGTAATACAC3′(SEQ ID NO 60), hybridized to the coding strand of the light chaincorresponding to the 3′ end of the framework 3 region. Theoligonucleotide primers were synthesized by Operon as described inExample 1A.

The PCR reaction was performed in a 100 ul reaction as described abovein Example 1A with the exception that 10 ng of template pC3AP313 wasused. The amplification cycles were performed as described in Example1A. The resultant PCR amplification products were then gel purified asdescribed in Example 1A and used in an overlap extension PCR reactionwith the products of the second PCR reaction, both as described below,to recombine the two products into reconstructed light chains containingmutagenized CDR3.

The second PCR reaction resulted in the amplification of the light chainfrom the 3′ end of framework region 3 extending to the end of CH1region. To amplify this region for encoding a 4 random amino acidresidue sequence in the CDR3, the following primer pairs were used. The5′ coding oligonucleotide primer pool, designated KV5R, had thenucleotide sequence represented by the formula, 5′TATTACTGTCAGCAGTATNNKNNKNNKNNKACTTTCGGCGGAGGGACCAAGG TGGAG3′ (SEQ ID NO61), where N can be A, C, G, or T and K is either G or T. The 3′noncoding primer, T7B, hybridized to the coding strand at the 3′ end ofCH1 having the sequence 5′ AATACGACTCACTATAGGGCG3′ (SEQ ID NO 62). The5′ end of the primer pool is complementary to the 3′ end of framework 3represented by the complementary nucleotide sequence of theoligonucleotide primer KV12B and the 3′ end of the primer pool iscomplementary to the 5′ end of framework 4. The region between the twospecified ends of the primer pool is represented by a 12-mer NNKdegeneracy. The second PCR reaction was performed on the pC3AP313 in a100 ul reaction as described above containing 1 ug of each ofoligonucleotide primers. The resultant PCR products encode a diversepopulation of mutagenized light chain CDR3 regions of 4 amino acidresidues in length with a conserved aspartic acid residue in the fourthamino acid residue position in the CDR3. The products were then gelpurified as described above.

One hundred nanograms of gel purified products from the first and secondPCR reactions were then admixed with 1 ug each of KEF and T7Boligonucleotide primers as a primer pair in a final PCR reaction to forma complete light chain fragment by overlap extension. The PCR reactionadmixture also contained 10 ul 10× PCR buffer, 1 ul TAQ POLYMERASE and 8ul 2.5 mM dNTP's as described above. The PCR reaction -was performed asdescribed in Example 1A.

To obtain sufficient quantities of amplification product, 15 identicalPCR reactions were performed. The resulting light chain fragmentsbeginning at framework 1 and extending to the end of CH1 and having arandomly mutagenized CDR3 region for encoding 4 amino acid residues. Thelight chain fragment amplification products from the 15 reactions werefirst pooled and then gel purified as described above prior to theirincorporation into the pc3AP313 surface display phagemid expressionvector to form a library as described in Example 2.

To create synthetic binding site sequences for encoding CDR3 regionshaving length of 5, 6 and 10 amino acid residues, the following primerswere used separately in the second PCR reaction in combination with the3′ primer, T7B: 1) for encoding a 5 amino acid sequence, the KV5R primerhad the formula 5′ TATTACTGTCAGCAGTATNNKNNKNNKNNKNNKACTTTCGGCGGAGGGACCAAGGTGGAG3′ (SEQ ID NO 61), where N is A, C, G or T and K is G or T; forencoding a 6 amino acid sequence, the KV6R primer had the formula 5′GATTTTGCAGTGTATTACTGTCAGCAGTATNNKNNKNNKNNKNNKNNKACTTTCGGCGGAGGGACCAAGGTGGAG3′ (SEQ ID NO 63),where N is A, C, G or T and Kis G or T; and for encoding a 10 amino acid sequence, the KV10OR primerhad the formula 5′ GATTTTGCAGTGTATTACTGTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKTTCGGCGGAGGGACCAAGGTGGAG3′ (SEQ ID NO 64), where N is A, C, G or T and Kis G or T.

The CDR3 light chain synthetic binding sites produced in the various PCRreactions are then introduced into libraries for screening. They can beused independently or in concert with similar binding site sequencescreated in the heavy chain CDR3 domain. The use of the two syntheticbinding site protein-encoding sequences enhances the ability ofobtaining synthetic binding site proteins displayed on human Fabs thatexhibit unexpected affinities and avidities to preselected targetmolecules. Thus, the synthetic binding site compositions in the heavyand light chain CDR3 domains of this invention allow for the productionof reactive binding molecules not normally attainable that havetherapeutic and diagnostic uses.

7. Deposit of Materials

The following cell lines and plasmids have been deposited on Feb. 2,1993, with the American Type Culture Collection, 10801 University Blvd.,Manassas, Va., USA (ATCC):

Material ATCC Accession No. E. coli harboring plasmid pMT12 ATCC 69079Plasmid pC3AP313 ATCC 75408 Plasmid p7EIII ATCC 75409

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The deposits will be made available by ATCC underthe terms of the Budapest Treaty which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 CFR §1.14 with particular reference to 886 OG 638). Theassignee of the present application has agreed that if the culturedeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain or plasmid is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines and plasmidsdeposited, since the deposited embodiment is intended as a singleillustration of one aspect of the invention and any cell lines orplasmid vectors that are functionally equivalent are within the scope ofthis invention. The deposit of material does not constitute an admissionthat the written description herein contained is inadequate to enablethe practice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustration that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A method for producing a polypeptide having a binding site that bindsa preselected antigen, the method comprising the steps of: i) amplifyinga complementarity determining region (CDR) encoding portion of atemplate immunoglobulin variable domain gene selected from the groupconsisting of a template immunoglobulin heavy chain variable domain geneand a template immunoglobulin light chain variable domain gene, whereinsaid template immunoglobulin heavy or light chain genes each have aframework encoding region and said CDR encoding portion and encoderespective heavy and light chain variable domain polypeptides having apreselected antigen binding specificity to a first antigen, and whereinsaid amplifying is by a primer extension reaction using a primerextension reaction oligonucleotide or its complement, for mutagenizing apreselected nucleotide in said CDR encoding portion, thereby forming alibrary of amplified CDR-mutagenized encoding immunoglobulin genefragments, said primer extension reaction oligonucleotide having a 3′and 5′ termini and comprising: a) a nucleotide sequence at the 3′terminus that hybridizes to a first framework encoding region of saidselected template immunoglobulin variable domain gene, said firstframework encoding region being located adjacent and 5′ to said CDRencoding portion; b) a nucleotide sequence at the 5′ terminus thathybridizes to a second framework encoding region of said selectedtemplate immunoglobulin variable domain gene, said second frameworkencoding region being located adjacent and 3′ to said CDR encodingportion; and c) a nucleotide sequence between said 5′ and 3′ terminiaccording to the formula:—X—[MNN]_(a)—Y—[MNN]_(b)—X—, where the sum of a and b is from 5 to 50, Xis a trinucleotide encoding cysteine or a native amino acid residuecoded by the immunoglobulin gene, N is independently any nucleotide, Mis adenine (A) or cytosine (C), Y is a nucleotide sequence that encodesa preselected polypeptide sequence of from 3 to 50 amino acid residues,Y includes the tripeptide RGD, and said 5′ and 3′ terminal nucleotidesequences have a length of about 6 to 50 nucleotides in length; ii)inserting individual members of the library of amplified CDR encodingportions formed in step (i) into a dicistronic phagemid expressionvector comprising immunoglobulin heavy and light chain variable domaingenes that lack the immunoglobulin gene portion corresponding to thefragment to be inserted, wherein upon insertion said vector is capableof expressing heavy and light chain variable domain polypeptides encodedby said vector, thereby forming a library of dicistronic expressionvectors containing amplified CDR-mutagenized immunoglobulin genefragments; iii) expressing said immunoglobulin heavy and light chaingenes in the library of dicistronic expression vectors formed in step(ii) in a host cell whereby said encoded heavy and light chain variabledomain polypeptides assemble on the surface of a phage to form aphage-displayed immunoglobulin heterodimer, thereby producing a libraryof CDR-mutagenized phage-displayed immunoglobulin heterodimers; and iv)immunoreacting members of the library of CDR-mutagenized phage-displayedimmunoglobulin heterodimers formed in step (iii) on a preselected secondantigen, said second antigen being different than said first antigen, toallow for selection of a CDR-mutagenized phage-displayed immunoglobulinheterodimer containing a polypeptide having a binding site capable ofbinding said preselected second antigen; wherein said binding site is anRGD-dependent binding site and wherein said oligonucleotide has theformula: 5′CTCCTCCTCCTCCTCGACGTCCATATAATAATT[MNN]_(a)ATCGCCACG[MNN]_(b)TGGCCCCACTCTCGCACAATAATA3′ (SEQ ID NO 49).
 2. A method for producing apolypeptide having a binding site that binds a preselected antigen, themethod comprising the steps of: i) amplifying a complementaritydetermining region (CDR) encoding portion of a template immunoglobulinvariable domain gene selected from the group consisting of a templateimmunoglobulin heavy chain variable domain gene and a templateimmunoglobulin light chain variable domain gene, wherein said templateimmunoglobulin heavy or light chain genes each have a framework encodingregion and said CDR encoding portion and encode respective heavy andlight chain variable domain polypeptides having a preselected antigenbinding specificity to a first antigen, and wherein said amplifying isby a primer extension reaction using a primer extension reactionoligonucleotide or its complement, for mutagenizing a preselectednucleotide in said CDR encoding portion, thereby forming a library ofamplified CDR-mutagenized encoding immunoglobulin gene fragments, saidprimer extension reaction oligonucleotide having a 3′ and 5′ termini andcomprising: a) a nucleotide sequence at the 3′ terminus that hybridizesto a first framework encoding region of said selected templateimmunoglobulin variable domain gene, said first framework encodingregion being located adjacent and 5′ to said CDR encoding portion; b) anucleotide sequence at the 5′ terminus that hybridizes to a secondframework encoding region of said selected template immunoglobulinvariable domain gene, said second framework encoding region beinglocated adjacent and 3′ to said CDR encoding portion; and c) anucleotide sequence between said 5′ and 3′ termini according to theformula:—X—[MNN]_(a)—Y—[MNN]_(b)—X—, where the sum of a and b is from 5 to 50, Xis a trinucleotide encoding cysteine or a native amino acid residuecoded by the immunoglobulin gene, N is independently any nucleotide, Mis adenine (A) or cytosine (C), Y is a nucleotide sequence that encodesa preselected polypeptide sequence of from 3 to 50 amino acid residues,Y includes the tripeptide RGD, and said 5′ and 3′ terminal nucleotidesequences have a length of about 6 to 50 nucleotides in length; ii)inserting individual members of the library of amplified CDR encodingportions formed in step (i) into a dicistronic phagemid expressionvector comprising immunoglobulin heavy and light chain variable domaingenes that lack the immunoglobulin gene portion corresponding to thefragment to be inserted, wherein upon insertion said vector is capableof expressing heavy and light chain variable domain polypeptides encodedby said vector, thereby forming a library of dicistronic expressionvectors containing amplified CDR-mutagenized immunoglobulin genefragments; iii) expressing said immunoglobulin heavy and light chaingenes in the library of dicistronic expression vectors formed in step(ii) in a host cell whereby said encoded heavy and light chain variabledomain polypeptides assemble on the surface of a phage to form aphage-displayed immunoglobulin heterodimer, thereby producing a libraryof CDR-mutagenized phage-displayed immunoglobulin heterodimers; and iv)immunoreacting members of the library of CDR-mutagenized phage-displayedimmunoglobulin heterodimers formed in step (iii) on a preselected secondantigen, said second antigen being different than said first antigen, toallow for selection of a CDR-mutagenized phage-displayed immunoglobulinheterodimer containing a polypeptide having a binding site capable ofbinding said preselected second antigen; wherein said binding site is anRGD-dependent binding site; and wherein said oligonucleotide has theformula: 5′CTCCTCCTCCTCCTCGACGTCCATATAATAGCA[MNN]_(a)ATCGCCACG[MNN]_(b)GCACCCCACTCTCGCACAATAATA3′ (SEQ ID NO 50).
 3. A method for producing apolypeptide having a binding site that binds a preselected antigen, themethod comprising the steps of: i) amplifying a complementaritydetermining region (CDR) encoding portion of a template immunoglobulinvariable domain gene selected from the group consisting of a templateimmunoglobulin heavy chain variable domain gene and a templateimmunoglobulin light chain variable domain gene, wherein said templateimmunoglobulin heavy or light chain genes each have a framework encodingregion and said CDR encoding portion and encode respective heavy andlight chain variable domain polypeptides having a preselected antigenbinding specificity to a first antigen, and wherein said amplifying isby a primer extension reaction using a primer extension reactionoligonucleotide, or its complement, for mutagenizing a preselectednucleotide in said CDR encoding portion, thereby forming a library ofamplified CDR-mutagenized encoding immunoglobulin gene fragments, saidprimer extension reaction oligonucleotide having a 3′, and 5′ terminiand comprising: a) a nucleotide sequence at the 3′ terminus thathybridizes to a first framework region of said selected templateimmunoglobulin variable domain gene, said first framework region beinglocated adjacent and 5′ to said CDR encoding portion; b) a nucleotidesequence at the 5′ terminus that hybridizes to a second frameworkencoding region of said selected template immunoglobulin variable domaingene, said second framework encoding region being located adjacent and3′ to said CDR encoding portion; and c) nucleotide sequence between said5′ and 3′ termini according to the formula:-L-[MNN]₄—P— where L and P are each one to ten trinucleotides encodingpreselected CDR sequences, N is independently any nucleotide, M isadenine (A) or cytosine (C) or analogs thereof; ii) inserting individualmembers of the library of amplified CDR encoding portions formed in step(i) into a dicistronic phagemid expression vector comprisingimmunoglobulin heavy and light chain variable domain genes that lack theimmunoglobulin gene portion corresponding to the fragment to beinserted, wherein upon insertion said vector is capable of expressingheavy and light chain variable domain polypeptides encoded by saidvector, thereby forming a library of dicistronic expression vectorscontaining amplified CDR-mutagenized immunoglobulin gene fragments; iii)expressing said immunoglobulin heavy and light chain genes in thelibrary of dicistronic expression vectors formed in step (ii) in a hostcell whereby said encoded heavy and light chain variable domainpolypeptides assemble on the surface of a phage to form aphage-displayed immunoglobulin heterodimer, thereby producing a libraryof CDR-mutagenized phage-displayed immunoglobulin heterodimers; and iv)immunoreacting members of the library of CDR-mutagenized phage-displayedimmunoglobulin heterodimers formed in step (iii) on a preselected secondantigen, said second antigen being different than said first antigen, toallow for selection of a CDR-mutagenized phage-displayed immunoglobulinheterodimer containing a polypeptide having a binding site capable ofbinding said preselected second antigen; wherein said oligonucleotidehas the formula: 5′ CTCCTCCTCCTCCTCGACGTCCATATAATAGCAATTCCT[MNN]₄CCCAAACGAGCACCCCAC TCTCGCACAATAATA3′ (SEQ ID NO 52).